Language：English  

In our previous study, we identified multifunctional cationic peptides from enzymatic hydrolysates of rice bran proteins (RBPs) that have antimicrobial and lipopolysaccharide-neutralizing activities. In this study, we investigated the potential of the peptides RBP-LRR, RBP-EKL, and RBP-SSF to promote proliferation, angiogenesis (tube formation), and migration in human umbilical vein endothelial cells (HUVECs). To determine mechanisms of wound healing actions, angiogenic and migration-promoting activities of these peptides were evaluated following pretreatments of HUVECs with specific inhibitors. In these experiments, the cationic peptides RBP-LRR, RBP-EKL, and RBP-SSF induced cell proliferation at low concentrations of 0.1 μM or 1 μM. Moreover, the three cationic peptides had angiogenic activities at concentrations more than 1 μM in tube formation assays, and their effects were similar to those of LL-37. Subsequent scratch migration assays exhibited that RBP-LRR, RBP-EKL, and RBP-SSF promote wound closure at optimum concentrations of 10, 10, and 0.1 μM, respectively. In further studies, we performed tube formation assays using HUVECs pretreated with SU5416, which inhibits vascular endothelial growth factor (VEGF) receptors, and suggested the possibility that the three cationic peptides induce angiogenesis by activating VEGF receptors. In corresponding scratch migration assays using HUVECs, pretreatment with the proliferation inhibitor mitomycin C did not alter the effects of RBP-LRR and RBP-EKL, and significant contribution to wound closure were mediated by cell migration regardless of proliferation rates. In contrast, RBP-SSF contributed to wound closure exclusively by promoting cell proliferation. The present data indicate that RBP-LRR, RBP-EKL, and RBP-SSF are candidates for use as wound healing agents.

DOI： 10.1016/j.jbiosc.2019.02.002

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DOI： 10.1016/j.heliyon.2019.e01490

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Language：English  

In this study, we investigated the lipopolysaccharide (LPS)-neutralizing and angiogenic activities of cationic peptides derived from the traditional Japanese fermented product Natto, which is made by fermenting cooked soybeans using Bacillus subtilis. Initially, we prepared 20 fractions of Natto extracts with various isoelectric points (pI's) using ampholyte-free isoelectric focusing (autofocusing). Cationic peptides were then purified from fractions 19 and 20, whose pH values were greater than 12, using reversed-phase high-performance liquid chromatography, and were identified using matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. Among the 13 identified cationic peptides, seven (KFNKYGR, FPFPRPPHQK, GQSSRPQDRHQK, QRFDQRSPQ, ERQFPFPRPPHQK, GEIPRPRPRPQHPE, and EQPRPIPFPRPQPR) had pI's greater than 9.5, positive net charges, and differing molecular weights. These peptides were then chemically synthesized and applied to chromogenic LPS-neutralizing assays using Limulus amebocyte lysates, and 50% effective (neutralizing) concentrations of 2.6-5.5 μM were demonstrated. In addition, tube formation assays in human umbilical vein endothelial cells revealed angiogenic activities for all but one (GEIPRPRPRPQHPE) of these seven cationic peptides, with increases in relative tube lengths of 23-31% in the presence of peptides at 10 μM. Subsequent experiments showed negligible hemolytic activity of these peptides at concentrations of up to 500 μM in mammalian red blood cells. Collectively, these data demonstrate that six cationic peptides from Natto extracts, with the exception of GEIPRPRPRPQHPE, have LPS-neutralizing and angiogenic activities but do not induce hemolysis.

DOI： 10.1016/j.jbiosc.2018.09.016

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Language：English  

In this study, we prepared fractions containing multifunctional cationic peptides by separating the commercial soybean protein hydrolysate Hinute-AM into 20 fractions. These fractions contained peptides with various isoelectric points (pI), as indicated by ampholyte-free isoelectric focusing (autofocusing). Thus, we purified and identified the cationic peptides from fractions 19 and 20, which had pH values greater than 10, using reversed-phase high-performance liquid chromatography and matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. Among 19 identified cationic peptides, NKNAKPPSPR, PGKKNAIV, KSGPGMSPR, NVSKPPRVV, RKVGAGGRKPLG, and LPCVIGGVPKRV had high pI values and were included as chemically synthesized peptides in assays of various functions, including lipopolysaccharide (LPS)-neutralizing and angiogenic activities. Chromogenic LPS-neutralizing assays using Limulus amebocyte lysates showed that 50% effective concentrations of these six peptides were between 1.63 and 2.65 μM, and were higher than that (0.12 μM) of polymyxin B. Moreover, in tube-formation assays in human umbilical vein endothelial cells, all of the six cationic peptides except LPCVIGGVPKRV exhibited angiogenic activities similar to those of the positive control LL-37. In addition, the six identified cationic peptides had no hemolytic activity at concentrations up to 500 μM in mammalian red blood cells. Our results demonstrate that five of the identified cationic peptides, excluding LPCVIGGVPKRV, have multiple functions and little or no hemolytic activity. These data indicate that fractions containing cationic peptides from Hinute-AM have the potential to be used as dietary supplements and functional ingredients in food products.

DOI： 10.1016/j.jbiosc.2018.07.013

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Language：English   Publishing type：Research paper (scientific journal)  

OBJECTIVE: Food-derived peptides have been reported to exhibit antibacterial activity against periodontal pathogenic bacteria. However, no effect has been shown on inflammation and bone resorption in periodontal pathology. The overall objective of the current study was to investigate how rice peptides influence biological defense mechanisms against periodontitis-induced inflammatory bone loss, and identify their novel functions as a potential anti-inflammatory drug. DESIGN: The expression of inflammatory and osteoclast-related molecules was examined in mouse macrophage-derived RAW 264.7 cell cultures using qPCR. Subsequently, the effect of these peptides on inflammatory bone loss in mouse periodontitis was examined using a mouse model of tooth ligation. Briefly, periodontal bone loss was induced for 7 days in mice by ligating the maxillary second molar and leaving the contralateral tooth un-ligated (baseline control). The mice were microinjected daily with the peptide in the gingiva until the day before euthanization. One week after the ligation, TRAP-positive multinucleated cells (MNCs) were enumerated from five random coronal sections of the ligated sites in each mouse. RESULTS: Rice peptides REP9 and REP11 significantly inhibited transcription activity of inflammatory and osteoclast-related molecules. Local treatment with the rice peptides, in mice subjected to ligature-induced periodontitis, inhibited inflammatory bone loss, explaining the decreased numbers of osteoclasts in bone tissue sections. CONCLUSION: Therefore, these data suggested that the rice peptides possess a protective effect against periodontitis.

DOI： 10.1016/j.archoralbio.2018.11.021

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1252/jcej.18we084

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DOI： 10.1038/s41598-018-29715-w

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In our previous studies, we showed that AmyI-1-18 and its single amino acid-substituted analogs have antimicrobial, anti-inflammatory, and anti-endotoxic activities and cause little or no hemolysis or cytotoxicity. In this study, we investigated the potential of these peptides to promote proliferation, angiogenesis (tube formation), and migration in human umbilical vein endothelial cells (HUVECs). Among five single amino acid-substituted analogs, [N3L]AmyI-1-18 induced cell proliferation in a concentration-dependent manner with similar efficacy to AmyI-1-18. In tube formation assays, AmyI-1-18 and [N3L]AmyI-1-18 had angiogenic activities at 1 μM and their effects were similar to those of LL-37. Moreover, scratch migration assays showed that AmyI-1-18, [N3L]AmyI-1-18, and LL-37 promote cell migration with optimum concentrations of 10, 1, and 0.1 μM, respectively. Subsequently, we performed tube formation assays using HUVECs pretreated with SU5416, which is an inhibitor of vascular endothelial growth factor (VEGF) receptors, and revealed that AmyI-1-18 and [N3L]AmyI-1-18 induce angiogenesis by activating VEGF receptors. Similarly, after pretreating HUVECs with mitomycin C, which inhibits cell proliferation, [N3L]AmyI-1-18 significantly contributed to wound closure in scratch migration assays. Moreover, enhancements of hydrophobicity following substitution of AmyI-1-18 asparagine with leucine led to greater increases in cell migration. The present data indicate that both peptides, particularly [N3L]AmyI-1-18, are candidates for use as wound healing agents.

DOI： 10.1016/j.peptides.2018.04.017

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Ltd  

Objectives The NCBI gene database and human-transcriptome database for alternative splicing were used to determine the expression of mRNAs for P-B (SMR3B) and variant form of P-B. The translational product from the former mRNA was identified as the protein named P-B, whereas that from the latter has not yet been elucidated. In the present study, we investigated the expression of P-B and its variant form at the protein level. Design To identify the variant protein of P-B, (1) cationic proteins with a higher isoelectric point in human pooled whole saliva were purified by a two dimensional liquid chromatography
(2) the peptide fragments generated from the in-solution of all proteins digested with trypsin separated and analyzed by MALDI-TOF-MS
and (3) the presence or absence of P-B in individual saliva was examined by 15% SDS-PAGE. Results The peptide sequences (I37PPPYSCTPNMNNCSR52, C53HHHHKRHHYPCNYCFCYPK72, R59HHYPCNYCFCYPK72 and H60HYPCNYCFCYPK72) present in the variant protein of P-B were identified. The peptide sequence (G6PYPPGPLAPPQPFGPGFVPPPPPPPYGPGR36) in P-B (or the variant) and sequence (I37PPPPPAPYGPGIFPPPPPQP57) in P-B were identified. The sum of the sequences identified indicated a 91.23% sequence identity for P-B and 79.76% for the variant. There were cases in which P-B existed in individual saliva, but there were cases in which it did not exist in individual saliva. Conclusions The variant protein is produced by excising a non-canonical intron (CC-AC pair) from the 3′-noncoding sequence of the PBII gene. Both P-B and the variant are subject to proteolysis in the oral cavity.

DOI： 10.1016/j.archoralbio.2018.01.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

In this study, we hydrolyzed rice endosperm protein (REP) with pepsin and generated 20 fractions containing multifunctional cationic peptides with varying isoelectric point (pI) values using ampholyte-free isoelectric focusing (autofocusing). Subsequently, we determined antimicrobial activities of each fraction against the pathogens Prophyromonas gingivalis, Propionibacterium acnes, Streptocossus mutans, and Candida albicans. Fractions 18, 19, and 20 had pI values greater than 12 and exhibited antimicrobial activity against P. gingivalis, P. acnes, and C. albicans, but not against S. mutans. In further experiments, we purified and identified cationic peptides from fractions 18, 19, and 20 using reversed-phase high-performance liquid chromatography and matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. We also chemically synthesized five identified peptides (RSVSKSR, RRVIEPR, ERFQPMFRRPG, RVRQNIDNPNRADTYNPRAG, and VVRRVIEPRGLL) with pI values greater than 10.5 and evaluated antimicrobial, lipopolysaccharide (LPS)-neutralizing, and angiogenic activities. Among these synthetic peptides, only VVRRVIEPRGLL exhibited antimicrobial activity against P. gingivalis, with an IC50 value of 87 mu M. However, all five cationic peptides exhibited LPS-neutralizing and angiogenic activities with little or no hemolytic activity against mammalian red blood cells at functional concentrations. These present data show dual or multiple functions of the five identified cationic peptides with little or no hemolytic activity. Therefore, fractions containing cationic peptides from REP hydrolysates have the potential to be used as dietary supplements and functional ingredients in food products.

DOI： 10.1016/j.peptides.2017.09.019

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

In this study, to prepare the fraction containing multifunctional cationic peptides, we first hydrolyzed rice bran protein (RBP) with pepsin. We separated the enzymatic hydrolysate of RBP into 20 fractions containing peptides with different isoelectric point (pI) values by ampholyte-free isoelectric focusing (autofocusing). Subsequently, we examined the antimicrobial activity of each fraction against four pathogens. In addition, we purified the cationic peptides from fractions exhibiting antimicrobial activity by reversed phase high-performance liquid chromatography and identified them by matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. Of five cationic peptides identified, we chemically synthesized three peptides with high pI values and evaluated their multiple functions, including antimicrobial, lipopolysaccharide-neutralizing and angiogenic activities. Our results demonstrated that the three identified cationic peptides exhibited multiple functions with little or no haemolytic activity. Fractions containing cationic peptides obtained from RBP hydrolysate have the potential to be used as dietary supplements and functional ingredients in food products. (C) 2017 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2017.04.046

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

In this study, we identified and chemically synthesized three cationic and amphipathic peptides (Glycinin-17, BCAS-16, and BCBS-11) from soybean proteins. These peptides had high isoelectric points, high positive net charges, and included multiple hydrophobic amino acids. Subsequently, we identified multiple functions of these peptides, including antimicrobial, lipopolysaccharide-neutralizing, and angiogenic activities, and examined their cytotoxic activities against mammalian red blood cells. Glycinin-17, BCAS-16, and BCBS-11 exhibited antimicrobial activity against Porphyromonas gingivalis and Candida albicans whereas Glycinin-17 did not possess antimicrobial effects on Propionibacterium acnes and Streptococcus mutans. Membrane-depolarization assays and flow cytometric analyses showed that the antimicrobial properties of Glycinin-17, BCAS-16, and BCBS-11 against P. gingivalis, P. acnes, and S. mutans were dependent on membrane-disrupting potential. In contrast, major antimicrobial activities of these peptides against C. albicans were dependent on interactions with targets other than cell membranes. Furthermore, chromogenic Limulus amebocyte lysate assays showed that 50% effective concentrations (EC50, 0.12-0.31 mu M) of these three peptides neutralize LPS with similar potency (EC50: 0.11 mu M) to that of polymyxin B. Moreover, tube-formation assays in human umbilical vein endothelial cells showed similar angiogenic activities of the three peptides as that following treatment with LL-37. Although BCAS-16 exhibited hemolytic activity, the rate of hemolysis for Glycinin-17 and BCBS-11 in the presence of 500-mu M Glycinin-17 and BCBS-11 was less than 2%. These results demonstrate that cationic and amphipathic peptides from soybean proteins, particularly Glycinin-17 and BCBS-11, have potential as multifunctional ingredients for healthcare applications.

DOI： 10.1002/bip.23023

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Association for Oral Biology  

Background Extensive peptidomic studies of human saliva have resulted in considerable advances in the field of proteomics. As the next generation in salivary research, a comprehensive understanding of the biological functions of in vivo peptides generated by proteolysis in the oral cavity has been long awaited. A cyclopedic functional analysis of salivary peptides may bring promising therapeutic agents and novel clinical applications.Highlight: (1) This review article refers to bioactive peptides hidden in salivary parent proteins. (2) Functions of the peptides as anti-microbial, anti-viral, wound-closing, and anti-pain are described. (3) Biological significances of the repeated structures in salivary proline-rich proteins are emphasized. Conclusion Human salivary proteins have the ability to generate bioactive peptides upon proteolytic cleavage.

DOI： 10.1016/j.job.2016.11.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Previously, we showed that the antimicrobial cationic and amphipathic octadecapeptide AmyI-1-18 from rice a-amylase (AmyI-1) inhibited the endotoxic activity of lipopolysaccharide (LPS) from Escherichia coli. In addition, we demonstrated that several AmyI1-18 analogs containing arginine or leucine substitutions, which were designed on the basis of the helical wheel projection of AmyI-1-18, exhibited higher antimicrobial activity against human pathogenic microorganisms than AmyI-1-18. In the present study, anti-inflammatory (anti-endotoxic) activities of five AmyI-1-18 analogs containing arginine or leucine substitutions were investigated. Two single arginine-substituted and two single leucine-substituted AmyI-1-18 analogs inhibited the production of LPS-induced nitric oxide in mouse macrophages (RAW264) more effectively than AmyI-1-18. These data indicate that enhanced cationic and hydrophobic properties of AmyI-1-18 are associated with improved anti-endotoxic activity. In subsequent chromogenic Limulus amebocyte lysate assays, 50% inhibitory concentrations (IC50) of the three AmyI-1-18 analogs (G12R, D15R, and E9L) were 0.11-0.13 mu M, indicating higher anti-endotoxic activity than that of AmyI-1-18 (IC50, 0.22 mu M), and specific LPS binding activity. In agreement, surface plasmon resonance analyses confirmed direct LPS binding of three AmyI-1-18 analogs. In addition, AmyI-1-18 analogs exhibited little or no cytotoxic activity against RAW264 cells, indicating that enhancements of antiinflammatory and LPS-neutralizing activities following replacement of arginine or leucine did not result in significant increases in cytotoxicity. This study shows that the arginine-substituted and leucine-substituted AmyI-1-18 analogs with improved antiendotoxic and antimicrobial activities have clinical potential as dual-function host defense agents. Copyright (C) 2017 European Peptide Society and John Wiley & Sons, Ltd.

DOI： 10.1002/psc.2983

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

The antimicrobial peptide Amyl-1-18 is a cationic alpha-helical octadecapeptide derived from alpha-amylase in rice (Oryza sativa L. japonica) that contains four cationic amino acid residues (two arginines and two lysines). To enhance the antibacterial activity of Amyl-1-18 against Porphyromonas gingivalis (a bacterium associated with periodontal disease), we synthesized 12 analogs bearing substitutions with alanine, leucine, and/or arginine that were designed based on helical wheel projections and investigated their antibacterial properties. The antibacterial properties of four analogs bearing substitution of a single arginine or lysine with alanine were almost similar to those of Amyl-1-18, suggesting that the antibacterial properties depend on the presence of three cationic amino acid residues. Of three single arginine-substituted analogs, Amyl-1-18(G12R) exhibited an antibacterial activity 2.8-fold higher [50% growth-inhibitory concentration (IC50): 4.6 mu M] than that of Amyl-1-18 (IC50: 13 mu M). Likewise, the antibacterial properties of two single leucine-substituted analogs were significantly enhanced; in particular, Amyl-1-18(N3L) exhibited an antibacterial activity (IC50: 2.5 mu M) 5.2-fold higher than that of Amyl-1-18. The hemolytic activity of Amyl-1-18(N3L) against mammalian red blood cells was low (2% at 50 mu M). A membrane-depolarization assay using a membrane potential-sensitive fluorescent dye revealed that, similar to Amyl-1-18, the antibacterial activity of Amyl-1-18(N3L) was not dependent on its membrane-disrupting activity. Our results demonstrate that the antibacterial properties of Amyl-1-18 against P. gingivalis are significantly improved, without a significant increase in hemolytic activity, by replacing asparagine with leucine at position 3. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2016.05.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：THE MEMBRANE SOCIETY OF JAPAN  

Here, we prepared a poly(L–lactic acid) (PLLA) microporous membrane with open finger–like pores as a scaffold for tissue engineering by a nonsolvent–induced phase separation method with the aid of a surfactant. The increase of surfactant content in the polymer solution caused the mold (glass plate) side of the membrane to adopt an indented structure with open finger–like pores to enhance the internal surface area. Osteoblast–like cells inoculated on the indented side grew on and in the PLLA membrane scaffold to reach 2 ～ 3 times higher cell density per unit apparent area compared to that attained in monolayer cultures. The cells on the membrane deposited calcium compounds by osteoinduction with ascorbic acid 2–phosphate, dexamethasone, and β– glycerophosphate. The PLLA membrane with open finger–like pores will likely be useful as scaffolds to support the implantation of osteogenic cells in bone tissue engineering.

DOI： 10.5360/membrane.41.304

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Other Link： http://search.jamas.or.jp/link/ui/2017289540

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

In our previous study, we used a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on Escherichia coli lysate, for evaluating the inhibition of green fluorescent protein (GFP) synthesis by pyrrhocoricin. In this study, using an RTS, we evaluated the inhibition of GFP synthesis by AmyI-1-18, an antimicrobial octadecapeptide. We found that, similarly to pyrrhocoricin, Amyl-1-18 inhibited GFP synthesis in the RTS in a concentration-dependent manner. In addition, the blockage of transcription and translation steps in the RTS was individually estimated using RT-PCR after gene expression to determine the mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that the inhibition of GFP synthesis by AmyI-1-18 did not occur at the transcription step but rather at the translation step. Furthermore, we assessed the inhibition of DnaK-mediated refolding of chemically denatured luciferase by AmyI-1-18; Amyl-1-18 inhibited the protein folding activity of the ATP-dependent DnaK/DnaJ molecular chaperone system in a concentration-dependent manner. Surface plasmon resonance (SPR) analysis showed that Amyl-1-18 strongly bound to RNA with a ICD value of 1.4 x 10(-8) M but not to DNA and that Amyl-1-18 specifically bound to DnaK with a K-D value of 4.4 x 10(-8) M. These SPR analysis results supported the results obtained in both the RTS and the molecular chaperone system. These results demonstrated that both RNA and DnaK are most likely the target of AmyI-1-18 in the protein synthesis system. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2016.03.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Rice (Oryza sativa) is consumed as a staple food globally, and rice bran, the byproduct, is an unused biomass that is ultimately discarded as waste. Thus, in the present study, a technique for producing tyrosinase inhibitory peptides from rice bran protein (RBP) was developed. Simultaneous treatment of RBP with chymotrypsin and trypsin produced numerous peptides. Subsequently, six tyrosinase inhibitory peptides were isolated from the hydrolysate fractions in a multistep purification protocol, and their amino acid sequences were determined. Three of these peptides had a C-terminal tyrosine residue and exhibited significant inhibitory effects against tyrosinase-mediated monophenolase reactions. Furthermore, peptide CT-2 (Leu-Gln-Pro-Ser-His-Tyr) potently inhibited melanogenesis in mouse B16 melanoma cells without causing cytotoxicity, suggesting the potential of CT-2 as an agent for melanin-related skin disorder treatment. The present data indicate that RBP is a potent source of tyrosinase inhibitory peptides and that simultaneous treatment of RBP with chymotrypsin and trypsin efficiently produces these peptides.

DOI： 10.1021/acs.jnatprod.6b00449

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Tyrosinase, a rate-limiting enzyme in melanin biosynthesis, catalyzes the hydroxylation of L-tyrosine to 3,4-dihydroxy-L-phenylalanine (L-dopa) (monophenolase reaction) and the subsequent oxidation of L-dopa to L-dopaquinone (diphenolase reaction). Thus, tyrosinase inhibitors have been proposed as skin-lightening agents; however, many of the existing inhibitors cannot be widely used in the cosmetic industry due to their high cytotoxicity and instability. On the other hand, some tyrosinase inhibitory peptides have been reported as safe. In this study, we found that the peptide TH10, which has a similar sequence to the characterized inhibitory peptide P4, strongly inhibits the monophenolase reaction with a half-maximal inhibitory concentration of 102 mu M. Seven of the ten amino acid residues in TH10 were identical to P4; however, TH10 possesses one N-terminal tyrosine, whereas P4 contains three tyrosine residues located at its N-terminus, center, and C-terminus. Subsequent analysis using sequence-shuffled variants indicated that the tyrosine residues located at the N-terminus and center of P4 have little to no contribution to its inhibitory activity. Furthermore, docking simulation analysis of these peptides with mushroom tyrosinase demonstrated that the active tyrosine residue was positioned close to copper ions, suggesting that TH10 and P4 bind to tyrosinase as a substrate analogue. (C) 2015 The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2015.10.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Objective: CL(14-25), a dodecapeptide of cyanate lyase from rice, is a novel cationic a-helical antimicrobial peptide. In this study, we examined inhibitory ability of CL(14-25) against endotoxic activities of lipopolysaccharides (LPSs) from Escherichia coli and periodontal pathogenic Aggregatibacter actinomycetemcomitans.
Methods: Endotoxin-neutralizing activity of CL(14-25) was evaluated by inhibition to induction of cytokine and nitric oxide in human aortic endothelial cells (HAECs) and RAW264 mouse macrophage cells, respectively. Protective effect of CL(14-25) was determined in mice against lethal toxicity of LPS.
Results: IL-6 in HAECs was induced by stimulation with LPS preparations of A. actinomycetemcomitans and E. coli tested in this study, and addition of CL(14-25) to the medium caused inhibition of their induction in a dose-dependent manner. CL(14-25) inhibited NO induction in RAW264 cells by a smooth type LPS of E. coli O55:B5 and an Rc type LPS of E. coli J5 as well as lipid A of E. coli R515 in a dose-dependent manner. Simultaneous injection of E. coli O55:B5 LPS and CL(14-25) in BALB/c mice resulted in prevention of lethal toxicity of the former. The results of a Limulus amebocyte lysate assay and surface plasmon resonance analysis of interaction between CL(14-25) and E. coli LPS or lipid A showed that CL(14-25) specifically binds to a lipid A moiety of LPS.
Conclusion: The results of present study suggest that CL(14-25) has a potential to be used as a nutraceutical agent for periodontal therapy. (C) 2015 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.micpath.2015.08.011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Previous studies have shown that pyrrhocoricin, a proline-rich antimicrobial peptide (PrAMP), killed sensitive species in a dose-dependent manner by specifically binding to DnaK. Here, on the basis of the finding that DnaK-deficient Escherichia coli strains are susceptible to PrAMPs, we used pyrrhocoricin to investigate internal targets other than DnaK. Using conventional antibiotics (bleomycin, streptomycin, and fosfomycin) that have known modes of action, first, we validated the availability of an assay using a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on E. coli lysate, for evaluating inhibition of protein synthesis. We found that, similarly to bleomycin and streptomycin, pyrrhocoricin inhibited GFP synthesis in RTS in a concentration-dependent manner. In addition, blockage of transcription and translation steps in RTS was individually estimated using RT-PCR after gene expression to determine mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that this inhibition of GFP synthesis by pyrrhocoricin did not occur at the transcription step but rather at the translation step, in a manner similar to that of GFP synthesis by streptomycin, an inhibitor of the translation step by causing misreading of tRNA. These results suggest that RTS is a powerful assay system for determining if antimicrobial peptides inhibit protein synthesis and its transcription and/or translation steps. This is the first study to have shown that pyrrhocoricin inhibited protein synthesis by specifically repressing the translation step. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2015.09.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

AmyI-1-18, an antimicrobial peptide, is a cationic -helical octadecapeptide derived from -amylase of rice (Oryza sativa L. japonica) that contains four cationic amino acid residues (two arginines and two lysines). To enhance the antifungal activity of AmyI-1-18 against Candida albicans, 11 analogs bearing substitutions with alanine, leucine, and/or arginine, which were designed on the basis of the helical wheel projection of AmyI-1-18, were synthesized, and their antifungal activity was investigated. The antifungal activities of four analogs obtained by replacing arginine or lysine with alanine were significantly reduced. The results suggested that the cationic arginine and lysine residues in AmyI-1-18 are important for its antifungal activity. The antifungal activities of two single leucine-substituted analogs were not improved, but among three single arginine-substituted analogs, AmyI-1-18(D15R) had approximately a twofold higher antifungal activity [50% growth-inhibitory concentration (IC50): 31 M] than AmyI-1-18 (IC50: 64 M) and exhibited low hemolytic activity (4% at 100 M). Flow cytometric analysis using propidium iodide revealed that the antifungal activity of AmyI-1-18(D15R) was dependent on its membrane-disrupting activity in a manner different from that of AmyI-1-18. Further enhancement of the cationicity and hydrophobicity of AmyI-1-18(D15R) resulted in no improvement in antifungal activity and a significant increase in hemolytic activity. In this study, the results demonstrated that the antifungal activity of AmyI-1-18 against C. albicans was enhanced through increasing its membrane-disrupting activity by replacing aspartic acid at position 15 with arginine without a significant increase in hemolytic activity. (c) 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 219-229, 2016.

DOI： 10.1002/bip.22817

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

We have previously reported that AmyI-1-18, an octadecapeptide derived from alpha-amylase (AmyI-1) of rice, is a novel cationic alpha-helical peptide that exhibited antimicrobial activity against human pathogens, including Porphyromonas gingivalis, Pseudomonas aeruginosa, Prop ionibacterium acnes, Streptococcus mutans, and Candida albicans. In this study, to further investigate the potential functions of AmyI-1-18, we examined its inhibitory ability against the endotoxic activities of lipopolysaccharides (LPSs, smooth and Rc types) and lipid A from Escherichia coli. AmyI-1-18 inhibited the production of endotoxin-induced nitric oxide (NO), an inflammatory mediator, in mouse macrophages (RAW264) in a concentration-dependent manner. The results of a chromogenic Limulus amebocyte lysate assay illustrated that the ability [50% effective concentration (EC50): 0.17 mu M] of AmyI-1-18 to neutralize lipid A was similar to its ability (EC50: 0.26 mu M) to neutralize LPS, suggesting that AmyI-1-18 specifically binds to the lipid A moiety of LPS. Surface plasmon resonance analysis of the interaction between AmyI-1-18 and LPS or lipid A also suggested that AmyI-1-18 directly binds to the lipid A moiety of LPS because the dissociation constant (K-D) of AmyI-1-18 with lipid A is 5.6 x 10(-1)0 M, which is similar to that (4.3 x 10(-10) M) of AmyI-1-18 with LPS. In addition, AmyI-1-18 could block the binding of LPS-binding protein to LPS, although its ability was less than that of polymyxin B. These results suggest that AmyI-1-18 expressing antimicrobial and endotoxin-neutralizing activities is useful as a safe and potent host defense peptide against pathogenic Gram-negative bacteria in many fields of healthcare. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.peptides.2015.11.006

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Azurin and Laz (lipidated azurin) are 2 bacterial proteins with anticancer, anti-viral and anti-parasitic activities. Azurin, isolated from the bacterium Pseudomonas aeruginosa, termed Paz, demonstrates anticancer activity against a range of cancers but not against brain tumors. In contrast, Laz is produced by members of Gonococci/Meningococci, including Neisseria meningitides which can cross the blood-brain barrier to infect brain meninges. It has been previously reported that Laz has an additional 39 amino acid moiety, called an H.8 epitope, in the N-terminal part of the azurin moiety that allows Laz to cross the entry barrier to brain tumors such as glioblastomas. Exactly, how the H.8 epitope helps the azurin moiety of Laz to cross the entry barriers to attack glioblastoma cells is unknown. In this paper, we describe the structural features of the H.8 moiety in Laz using X-ray crystallography and demonstrate that while the azurin moiety of Laz adopts a beta-sandwich fold with 2 beta-sheets arranged in the Greek key motif, the H.8 epitope was present as a disordered structure outside the Greek key motif. Structures of Paz and H.8 epitope-deficient Laz are well superimposed. The structural flexibility of the H.8 motif in Laz explains the extracellular location of Laz in Neisseria where it can bind the key components of brain tumor cells to disrupt their tight junctions and allow entry of Laz inside the tumors to exert cytotoxicity.

DOI： 10.1080/21655979.2015.1022303

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Microfiltration membranes of poly(L-lactic acid) (PLEA) have been prepared by a nonsolvent-induced phase separation method with the aid of surfactants. Surfactants with hydrophilic-lipophilic balance (HLB) values of 14.9-15.6 were found to be useful in reducing the shrinkage in thickness of the PLEA membrane. Among the surfactants examined, Tween 80 was the best for preparing microfiltration membranes. The surfactant allowed instantaneous phase separation and seemed to enhance the diffusion of water in the PLEA solution during structure formation. The membrane had asymmetric finger-like structures and showed low membrane resistance and high bacterial cell retention when the membrane was prepared from a 10 wt% PLEA solution in 1,4-dioxane containing 10 wt.% Tween 80. Bovine serum albumin molecules passed through the membrane suggesting that the membrane functions as a microfiltration membrane. The membrane was stable at 25 degrees C but degradable at 60 degrees C in wet conditions. The membrane can be applied as a compostable microfiltration membrane in food and biochemical industries. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.memsci.2015.01.021

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Amyl-1-18, an octadecapeptide derived from a-amylase (Amyl-1) of rice (Oryza sativa L. japonica), is a novel cationic a-helical antimicrobial peptide (AMP) that contains two lysine and two arginine residues. The antimicrobial activity of Amyl-1-18 against human pathogens was quantitatively evaluated using a chemiluminescence method that measures ATP derived from viable cells. Of the ten kinds of human pathogens, Amyl-1-18 exhibited antimicrobial activity against nine. Its 50% growth-inhibitory concentrations (ICs50) against Porphyromonas gingivalis, Propionibacterium acnes, Pseudomonas aeruginosa, Candida albicans, and Streptococcus mutans were 13, 19, 50, 64, and 77 mu M, respectively. Amyl-1-18 had little or no hemolytic activity even at 500 mu M, and showed negligible cytotoxicity up to 1200 mu M. The degree of 3,3'-dipropylthiadicarbocyanine iodide release from P. gingivalis cells induced by the addition of Amyl-1-18 was significantly lower than that induced by the addition of melittin. Flow cytometric analysis showed that the percentages of P. aeruginosa, S. mutans, and C. albicans cells stained with propidium iodide (PI), a DNA-intercalating dye, were 89%, 43%, and 3%, respectively, when Amyl-1-18 was added at a concentration equal to its 43IC(50). Therefore, the antimicrobial activity of Amyl-1-18 against P. aeruginosa and S. mutans appears to be mainly attributable to its membrane-disrupting activity. In contrast, its antimicrobial activity against P. gingivalis and C. albicans most likely depends upon interactions with intracellular targets other than their cell membranes. Collectively, these results indicate that Amyl-1-18 is useful as a safe and potent AMP against the pathogens described above in many fields of healthcare. (C) 2015 Wiley Periodicals, Inc.

DOI： 10.1002/bip.22605

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CL(14-25), a dodecapeptide, exhibits antimicrobial activity against Porphyromonas gingivalis with the 50% growth-inhibitory concentration (IC50) value of 145 mu M, and arginine-specific gingipain (Rgp)-inhibitory activity. Kinetic analysis revealed that CL(14-25) is a mixed-type inhibitor, with inhibition constants (K-i and K-i' values) of 1.4 x 10(-6) M and 4.3 x 10(-6) M, respectively. To elucidate the contributions of four cationic amino acid residues at the N- and C-termini of CL(14-25) toward Rgp-inhibitory activity, we investigated the Rgp-inhibitory activities of truncated and alanine-substituted analogs of CL(14-25). Rgp-inhibitory activities significantly decreased by truncated analogs, CL(15-25) and CL(1625), whereas those of CL(14-24) and CL(14-23) were almost as high as that of CL(14-25). Rgp-inhibitory activities of alanine-substituted analogs, CL(R14A) and CL(R14A, R15A) also significantly decreased, whereas those of CL(K25A) and CL(R24A, K25A) were higher than that of CL(14-25). These results suggest that the arginine residue at position 15 substantially contributes to the Rgp-inhibitory activity and that the arginine residue at position 14 plays important roles in exerting Rgp-inhibitory activity. In this study, we demonstrated that CL(K25A) was a potent, dual function, peptide inhibitor candidate, exhibiting Rgp-inhibitory activity with Ki and K-i(l) of 9.6 x 10(-7) M and 1.9 x 10(-6) M, respectively, and antimicrobial activity against P. gingivalis with an IC50 value of 51 mM. (C) 2014 Wiley Periodicals, Inc.

DOI： 10.1002/bip.22525

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

AmyI-1 is an alpha-amylase from Oryza sativa (rice) and plays a crucial role in degrading starch in various tissues and at various growth stages. This enzyme is a glycoprotein with an N-glycosylated carbohydrate chain, a unique characteristic among plant alpha-amylases. In this study, we report the first crystal structure of AmyI-1 at 2.2-angstrom resolution. The structure consists of a typical (beta/alpha)(8)-barrel, which is well-conserved among most alpha-amylases in the glycoside hydrolase family-13. Structural superimposition indicated small variations in the catalytic domain and carbohydrate-binding sites between AmyI-1 and barley alpha-amylases. By contrast, regions around the N-linked glycosylation sites displayed lower conservation of amino acid residues, including Asn-263, Asn-265, Thr-307, Asn-342, Pro-373, and Ala-374 in AmyI-1, which are not conserved in barley alpha-amylases, suggesting that these residues may contribute to the construction of the structure of glycosylated AmyI-1. These results increase the depths of our understanding of the biological functions of AmyI-1.

DOI： 10.1080/09168451.2014.917261

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Background and Objective: Porphyromonas gingivalis is a major etiological agent in the development and progression of periodontal diseases. In this study, we isolated a cell growth inhibitor against P. gingivalis species from rice protein extract.
Material and Methods: The cell growth inhibitor active against P. gingivalis was purified from polished rice extract using a six-step column chromatography process. Its antimicrobial properties were investigated through microscope analysis, spectrum of activity and general structure.
Results: The inhibitor was identified as AmyI-1, an alpha-amylase, and showed significant cell growth inhibitory activity against P. gingivalis species. Scanning electron microscopy micrograph analysis and bactericidal assay indicated an intriguing possibility that the inhibitor compromises the cell membrane structure of the bacterial cells and leads to cell death. Moreover, alpha-amylases from human saliva and porcine pancreas showed inhibitory activity similar to that of AmyI-1.
Conclusions: This is the first study to report that alpha-amylases cause cell death of periodontal pathogenic bacteria. This finding highlights the potential importance and therapeutic potential of alpha-amylases in treating periodontal diseases.

DOI： 10.1111/jre.12079

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The antimicrobial activity of analogs obtained by substituting arginine and lysine in CL(14-25), a cationic alpha-helical dodecapeptide, with alanine against Porphyromonas gingivalis, a periodontal pathogen, varied significantly depending on the number and position of cationic amino acids. The alanine-substituted analogs had no hemolytic activity, even at a concentration of 1 mM. The antimicrobial activities of CL(K20A) and CL(K20A, K25A) were 3.8-fold and 9.1-fold higher, respectively, than that of CL(14-25). The antimicrobial activity of CL(R15A) was slightly lower than that of CL(14-25), suggesting that arginine at position 15 is not essential but is important for the antimicrobial activity. The experiments in which the alanine-substituted analogs bearing the replacement of arginine at position 24 and/or lysine at position 25 were used showed that arginine at position 24 was crucial for the antimicrobial activity whenever lysine at position 25 was substituted with alanine. Helical wheel projections of the alanine-substituted analogs indicate that the hydrophobicity in the vicinity of leucine at position 16 and alanines at positions 18 and/or 21 increased by substituting lysine at positions 20 and 25 with alanine, respectively. The degrees of diSC(3)-5 release from P. gingivalis cells and disruption of GUVs induced by the alanine-substituted analogs with different positive charges were not closely related to their antimicrobial activities. The enhanced antimicrobial activities of the alanine-substituted analogs appear to be mainly attributable to the changes in properties such as hydrophobicity and amphipathic propensity due to alanine substitution and not to their extents of positive charge (cationicity). (C) 2013 Wiley Periodicals, Inc.

DOI： 10.1002/bip.22399

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

CL(14-25), a dodecapeptide, that is a partial region near N-terminus of cyanate lyase (CL, EC 4.3.99.1) from rice (Oryza sativa L. japonica), contains three arginine and two lysine residues. It was a novel cationic alpha-helical antimicrobial peptide. The antimicrobial activity of CL(14-25) against Porphyromonas gingivalis, a periodontal pathogen, was quantitatively evaluated by a chemiluminescence method that measures ATP derived from viable cells. The 50% growth-inhibitory concentration of CL(14-25) against P. gingivalis cells was 145 mu M. CL(14-25), even at a concentration of 800 mu M, had no hemolytic activity. When giant unilamellar vesicles (GUVs) that mimic the membrane composition of Gram-negative bacteria were used, microscopy image analysis suggested that CL(14-25) disrupted GUVs in a detergent-like manner. Therefore, CL(14-25) appears to exhibit antimicrobial activity through membrane disruption. To investigate the contribution of cationic amino acid residues in CL(14-25) to its antimicrobial activity, we synthesized four truncated CL analogs, in which one or two cationic amino acid residues were deleted from the N- and C- termini of CL(14-25). The degrees of calcein leakage from large unilamellar vesicles (LUVs) and 3,3'-dipropylthiadicarbocyanine iodide (diSC(3)-5) release from P. gingivalis cells induced by truncated CL analogs were closely related to their antimicrobial activities. CL analogs, which were truncated by removing an arginine residue from the N-terminus and a lysine residue from the C-terminus maintained their antimicrobial activity. However, CL analogs, which were further truncated by removing two arginine residues from the N-terminus, and an arginine and a lysine residue from the C-terminus, rarely exhibited antimicrobial activity. (c) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.peptides.2012.12.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Inc.  

Hsp70(241-258), an octadecapeptide derived from the heat shock protein 70 (Hsp70) of rice (Oryza sativa L. japonica), is a novel cationic β-helical antimicrobial peptide (AMP) that contains four lysine, two arginine, and two histidine residues. The antimicrobial activity of Hsp70(241-258) against Porphyromonas gingivalis, a periodontal pathogen, and Candida albicans, an opportunistic fungal pathogen, was quantitatively evaluated using a chemiluminescence method that measures ATP derived from viable cells. The 50% growth-inhibitory concentrations of Hsp70(241-258) against P. gingivalis and C. albicans cells were 63μM and 70μM, respectively. Hsp70(241-258) had little or no hemolytic activity even at 1 mM, and showed negligible cytotoxicity up to 300M. The degrees of calcein leakage from large unilamellar vesicles, which mimic the membranes of Gram-negative bacteria, and 3,3 β- dipropylthiadicarbocyanine iodide release from P. gingivalis cells induced by the addition of Hsp70(241-258) increased in a concentrationdependent manner. When Hsp70(241-258) was added to calcein-acetoxymethyl ester-loaded C. albicans cells, calcein release from the cells increased in a concentration-dependent manner. Flow cytometric analysis also showed that the percentages of C. albicans cells stained with propidium iodide, a DNAintercalating dye, increased as the concentration of Hsp70(241-258) added was increased. Therefore, Hsp70(241-258) appears to exhibit antimicrobial activity against P. gingivalis and C. albicans through membrane disruption. These results suggest that Hsp70(241-258) could be useful as a safe and potent AMP against P. gingivalis and C. albicans in many fields of health care, especially in the control of oral infections. © 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.peptides.2013.08.011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

NAD kinase catalyzes the phosphorylation of NAD(+) to synthesize NADP(+), whereas NADH kinase catalyzes conversion of NADH to NADPH. The mitochondrial protein Pos5 of Saccharomyces cerevisiae shows much higher NADH kinase than NAD kinase activity and is therefore referred to as NADH kinase. To clarify the structural determinant underlying the high NADH kinase activity of Pos5 and its selectivity for NADH over NAD(+), we determined the tertiary structure of Pos5 complexed with NADH at a resolution of 2.0 angstrom. Detailed analysis, including a comparison of the tertiary structure of Pos5 with the structures of human and bacterial NAD kinases, revealed that Arg-293 of Pos5, corresponding to His-351 of human NAD kinase, confers a positive charge on the surface of NADH-binding site, whereas the corresponding His residue does not. Accordingly, conversion of the Arg-293 into a His residue reduced the ratio of NADH kinase activity to NAD kinase activity from 8.6 to 2.1. Conversely, simultaneous changes of Ala-330 and His-351 of human NAD kinase into Ser and Arg residues significantly increased the ratio of NADH kinase activity to NAD kinase activity from 0.043 to 1.39; human Ala-330 corresponds to Pos5 Ser-272, which interacts with the side chain of Arg-293. Arg-293 and Ser-272 were highly conserved in Pos5 homologs (putative NADH kinases), but not in putative NAD kinases. Thus, Arg-293 of Pos5 is a major determinant of NADH selectivity. Moreover, Ser-272 appears to assist Arg-293 in achieving the appropriate conformation.

DOI： 10.1074/jbc.M111.249011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit. Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase. imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10 angstrom resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.01.043

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In Sphingomonas sp. A1, alginate is degraded by alginate lyases to its constituent monosaccharides, which are nonenzymatically converted to an alpha-keto acid, namely, 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). The properties of the DEH-metabolizing enzyme and its gene in strain A1 were characterized. In the presence of alginate, strain A1 cells inducibly produced an NADPH-dependent DEH reductase (A1-R) in their cytoplasm. Molecular cloning of the enzyme gene indicated that A1-R belonged to the short-chain dehydrogenase/reductase superfamily and catalyzed the conversion of DEH to 2-keto-3-deoxy-D-gluconic acid most efficiently at around pH 7.0 and 50 degrees C. Crystal structures of A1-R and its complex with NADP were determined at around 1.6 angstrom resolution by X-ray crystallography. The enzyme consists of three layers (alpha/beta/alpha) , with a coenzyme-binding Rossmann fold. NADP is surrounded by positively charged residues, and Gly-38 and Arg-39 are crucial for NADP binding. Site-directed mutagenesis studies suggest that Ser-150, Tyr-164, and Lys-168 located around the Rossmann fold constitute the catalytic triad. To our knowledge, this is the first report on molecular cloning and structure determination of a bacterial DEH reductase responsible for alginate metabolism. (C) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbapap.2010.05.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Alginate, a major component of the cell wall matrix in brown seaweeds, is degraded by alginate lyases through a beta-elimination reaction. Almost all alginate lyases act endolytically on substrate, thereby yielding unsaturated oligouronic acids having 4-deoxy-L-erythro-hex-4-enepyranosyluronic acid at the nonreducing end. In contrast, Agrobacterium tumefaciens alginate lyase Atu3025, a member of polysaccharide lyase family 15, acts on alginate polysaccharides and oligosaccharides exolytically and releases unsaturated monosaccharides from the substrate terminal. The crystal structures of Atu3025 and its inactive mutant in complex with alginate trisaccharide (H531A/Delta GGG) were determined at 2.10- and 2.99-angstrom resolutions with final R-factors of 18.3 and 19.9%, respectively, by x-ray crystallography. The enzyme is comprised of an alpha/alpha-barrel + anti-parallel beta-sheet as a basic scaffold, and its structural fold has not been seen in alginate lyases analyzed thus far. The structural analysis of H531A/Delta GGG and subsequent site-directed mutagenesis studies proposed the enzyme reaction mechanism, with His(311) and Tyr(365) as the catalytic base and acid, respectively. Two structural determinants, i.e. a short alpha-helix in the central alpha/alpha-barrel domain and a conformational change at the interface between the central and C-terminal domains, are essential for the exolytic mode of action. This is, to our knowledge, the first report on the structure of the family 15 enzyme.

DOI： 10.1074/jbc.M110.125450

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

The flagellar protein FlgJ, a member of glycoside hydrolase family 73, has N- and C-terminal domains that are responsible for flagellar rod assembly and peptidoglycan hydrolysis, respectively. The crystal structure of the C-terminal domain of SPH1045 (SPH1045-C), the FlgJ from Sphingomonas sp. strain A1, showed a long cleft formed by two lobes, alpha and beta. In this study, seven site-specific mutants of residues in the cleft were prepared and analyzed. Enzyme activity was reduced most significantly in mutants E185A and Y281A, followed by E224A. A comparison of the crystal structure of the inactive mutant E185A with that of other related enzymes revealed that Glu185 is structurally reasonable as the proton donor and that Tyr281 is close to Glu185. Glu224 is, however, far from the catalytic site, which is inconsistent with the decreased activity exhibited by E224A. The structural flexibility of Glu224 and its neighboring residues observed in SPH1045-C may indicate that this region is able to change its conformation upon substrate binding.

DOI： 10.1002/jobm.200900249

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

Sphingomonas sp. A1, a Gram-negative bacterium, directly internalizes alginate macromolecules through a mouth-like pit that is present on its cell surface. The alginate-binding protein Algp7, which was found to be localized on the cell surface, contributes to the accumulation of alginate in the pit. Algp7 was crystallized at 293 K by means of the sitting- drop vapour-diffusion method with polyethylene glycol 3350 as a crystallizing agent. Preliminary X-ray analysis showed that the Algp7 crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.1, b = 98.0, c = 100.1 angstrom, and that it diffracted to 2.8 angstrom resolution.

DOI： 10.1107/S1744309109013669

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A saprophytic Bacillus subtilis secretes two types of rhamnogalacturonan (RG) lyases, endotype YesW and exotype YesX, which are responsible for an initial cleavage of the RG type I (RG-I) region of plant cell wall pectin. Polysaccharide lyase family 11 YesW and YesX with a significant sequence identity (67.8%) cleave glycoside bonds between rhamnose and galacturonic acid residues in RG-I through a beta-elimination reaction. Here we show the structural determinants for substrate recognition and the mode of action in polysaccharide lyase family 11 lyases. The crystal structures of YesW in complex with rhamnose and ligand-free YesX were determined at 1.32 and 1.65 A resolution, respectively. The YesW amino acid residues such as Asn(152), Asp(172), Asn(532), Gly(533), Thr(534), and Tyr(595) in the active cleft bind to rhamnose molecules through hydrogen bonds and van der Waals contacts. Other rhamnose molecules are accommodated at the noncatalytic domain far from the active cleft, revealing that the domain possibly functions as a novel carbohydrate-binding module. A structural comparison between YesW and YesX indicates that a specific loop in YesX for recognizing the terminal saccharide molecule sterically inhibits penetration of the polymer over the active cleft. The loop-deficient YesX mutant exhibits YesW-like endotype activity, demonstrating that molecular conversion regarding the mode of action is achieved by the addition/removal of the loop for recognizing the terminal saccharide. This is the first report on a structural insight into RG-I recognition and molecular conversion of exotype to endotype in polysaccharide lyases.

DOI： 10.1074/jbc.M807799200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Glycoside hydrolase (GH) categorized into family 73 plays an important role in degrading bacterial cell wall peptidoglycan. The flagellar protein FlgJ contains N- and C-terminal domains responsible for flagellar rod assembly and peptidoglycan hydrolysis, respectively. A member of family GH-73, the C-terminal domain (SPH1045-C) of FlgJ from Sphingomonas sp. strain A1 was expressed in Escherichia coli, purified, and characterized. SPH1045-C exhibited bacterial cell lytic activity most efficiently at pH 6.0 and 37 degrees C. The X-ray crystallographic structure of SPH1045-C was determined at 1.74 angstrom resolution by single-wavelength anomalous diffraction. The enzyme consists of two lobes, a and p. A deep cleft located between the two lobes can accommodate polymer molecules, suggesting that the active site is located in the cleft. Although SPH1045-C shows a structural homology with family GH-22 and GH-23 lysozymes, the arrangement of the nucleophile/base residue in the active site is specific to each peptidoglycan hydrolase. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2009.01.186

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Gram-negative Sphingomonas sp. strain A1 accumulates alginate in the cell surface pit and directly incorporates the polysaccharide into its cytoplasm through a 'superchannel'. A cell surface protein Algp7 (27 kDa) is inducibly expressed in the presence of alginate. Although the protein Algp7 was initially classified as a lipoprotein based on its primary structure, Algp7 purified from strain A1 cells did not possess a lipid moiety. Algp7 bound alginate efficiently at a neutral pH with a K-d of 3.6 x 10(-8) M, suggesting that the cell surface protein contributed to accumulation of alginate in the pit.

DOI： 10.1111/j.1574-6968.2008.01354.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Rhamnogalacturonan (RG) lyase produced by plant pathogenic and saprophytic microbes plays an important role in degrading plant cell walls. An extracellular RG lyase YesW from saprophytic Bacillus subtilis is a member of polysaccharide lyase family 11 and cleaves glycoside bonds in polygalacturonan as well as RG type-I through a beta-elimination reaction. Crystal structures of YesW and its complex with galacturonan disaccharide, a reaction product analogue, were determined at 1.4 and 2.5 angstrom resolutions with final R-factors of 16.4% and 16.6%, respectively. The enzyme is composed of an eight-bladed beta-propeller with a deep cleft in the center as a basic scaffold, and its structural fold has not been seen in polysaccharide lyases analyzed thus far. Structural analysis of the disaccharide-bound YesW and a site-directed mutagenesis study suggested that Arg-452 and Lys-535 stabilize the carboxyl group of the acidic polysaccharide molecule and Tyr-595 makes a stack interaction with the sugar pyranose ring. In addition to amino acid residues binding to the disaccharide, one calcium ion, which is coordinated by Asp-401, Glu-422, His-363, and His-399, may mediate the enzyme activity. This is, to our knowledge, the first report of a new structural category with a beta-propeller fold in polysaccharide lyases and provides structural insights into substrate binding by RG lyase.

DOI： 10.1074/jbc.M704663200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Plant cell wall degradation is a premier event when Bacillus subtilis, a typical saprophytic bacterium, invades plants. Here we show the degradation system of rhamnogalacturonan type I (RG-I), a component of pectin from the plant cell wall, in B. subtilis strain 168. Strain 168 cells showed a significant growth on plant cell wall polysaccharides such as pectin, polygalacturonan, and RG-I as a carbon source. DNA microarray analysis indicated that three gene clusters (yesOPQRSTUVWXYZ, ytePQRST, and ybcMOPST-ybdABDE) are inducibly expressed in strain 168 cells grown on RG-I. Cells of an industrially important bacterium, B. subtilis strain natto, fermenting soybeans also express the gene cluster including the yes series during the assimilation of soybean used as a carbon source. Among proteins encoded in the yes cluster, YesW and YesX were found to be novel types of RG lyases releasing disaccharide from RG-I. Genetic and enzymatic properties of YesW and YesX suggest that strain 168 cells secrete YesW, which catalyzes the initial cleavage of the RG-I main chain, and the resultant oligosaccharides are converted to disaccharides through the extracellular exotype YesX reaction. The disaccharide is finally degraded into its constituent monosaccharides through the reaction of intracellular unsaturated galacturonyl hydrolases YesR and YteR. This enzymatic route for RG-I degradation in strain 168 differs significantly from that in plant-pathogenic fungus Aspergillus aculeatus. This is, to our knowledge, the first report on the bacterial system for complete RG-I main chain degradation.

DOI： 10.1128/AEM.00147-07

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The Gram-negative bacterium Sphingomonas sp. strain A I (strain A 1) has a peculiar biosystem to directly import and depolymerize a macromolecule, alginate, which is encoded by a cluster of genes on the genome. We identified five clustered ORFs homologous to some genes of the strain Al cluster in the genome of Agrobacterium tumefaciens strain C58 (strain C58). These ORFs are Atu3021, Am3022, Atu3023, and Atu3024, encoding a putative sugar ABC transporter system and Atu3025, which encodes a putative alginate lyase. We analyzed the involvement of this gene cluster in alginate metabolism. Strain C58 cells grew significantly on low-molecular-weight (LMW) alginate (average molecular weight, 1000), and we detected specific alginate-induced expression of Atu3024 and Atu3025. This strain does not grow on alginate (average molecular weight, 25 600), suggesting that the strain C58 gene cluster is involved in importing and degrading LMW alginate. One protein, Atu3025, purified from strain C58, was identified as an alginate lyase, and the enzyme overexpressed in Escherichia coli was further characterized. Atu3025 released monosaccharides specifically from alginate most efficiently at pH 7.3 and 30 degrees C through a beta-elimination reaction, indicating that Atu3025 is an exotype alginate lyase potentially involved in the assimilation of LMW alginate in strain C58. (c) 2006 Elsevier SAS. All rights reserved.

DOI： 10.1016/j.resmic.2006.02.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Bacillus subtilis strain 168 YteR has been identified as a novel enzyme "unsaturated rhamnogalacturonyl hydrolase" classified in glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) produced from plant cell wall RG type-I treated with RG lyases, releasing unsaturated galacturonic acid (Delta GalA) from the substrate. The most likely candidate catalytic residue is Asp-143. Here, we show the structure of D143N in complex with unsaturated RG disaccharide (substrate) determined at 1.9 angstrom resolution by X-ray crystallography. This structural feature directly contributes to the postulation of the enzyme reaction mechanism. YteR triggers the hydration of vinyl ether group in Delta GalA, but not of glycoside bond, by using Asp-143 as a general acid and base catalyst. Asp-143 donates proton to the double bond of Delta GalA as an acid catalyst and also deprotonates a water molecule as a base catalyst. Deprotonated water molecule attacks the C5 atom of Delta GalA. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2006.07.034

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

YteR, a hypothetical protein with unknown functions, is derived from Bacillus subtilis strain 168 and has an overall structure similar to that of bacterial unsaturated glucuronyl hydrolase (UGL), although it exhibits little amino acid sequence identity with UGL. UGL releases unsaturated glucuronic acid from glycosaminoglycan treated with glycosaminoglycan lyases. The amino acid sequence of YteR shows a significant homology (26% identity) with the hypothetical protein YesR also from B. subtilis strain 168. To clarify the intrinsic functions of YteR and YesR, both proteins were overexpressed in Escherichia coli, purified, and characterized. Based on their gene arrangements in genome and enzyme properties, YteR and YesR were found to constitute a novel enzyme activity, "unsaturated rhamnogalacturonyl hydrolase," classified as new glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) obtained from RG type-l treated with RG lyases and releases an unsaturated galacturonic acid. The crystal structure of YteR complexed with unsaturated chondroitin disaccharide (UGL substrate) was obtained and compared to the structure of UGL complexed with the same disaccharide. The UGL substrate is sterically hindered with the active pocket of YteR. The protruding loop of YteR prevents the UGL substrate from being bound effectively. The most likely candidate catalytic residues for general acid/ base are Asp143 in YteR and Asp135 in YesR. This is supported by three-dimensional structural and site-directed mutagenesis studies. These findings provide molecular insights into novel enzyme catalysis and sequential reaction mechanisms involved in RG-I depolymerization by bacteria. (c) 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2006.04.047

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Language：English   Publishing type：Research paper (scientific journal)  

Almost all alginate lyases depolymerize alginate in an endolytical fashion via a β-elimination reaction. The alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, consisting of 776 amino-acid residues, is a novel exotype alginate lyase classified into polysaccharide lyase family 15. The enzyme was crystallized at 293 K by sitting-drop vapour diffusion with polyethylene glycol 4000 as a precipitant. Preliminary X-ray analysis showed that the Atu3025 crystal belonged to space group P21 and diffracted to 2.8 Å resolution, with unit-cell parameters a = 107.7, b = 108.3, c = 149.5 Å, β= 91.5°. © 2006 International Union of Crystallography. All rights reserved.

DOI： 10.1107/S1744309106014333

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Rhamnogalacturonan lyases degrade rhamnogalacturonan I, a major component of pectin, through a beta-elimination reaction. YesW from Bacillus subtilis strain 168 is a novel rhamnogalacturonan lyase classified into polysaccharide lyase family 11 (PL-11). The enzyme was crystallized at 293 K using the sitting-drop vapour-diffusion method with 2-methyl-2,4-pentanediol (MPD) as a precipitant. Preliminary X-ray analysis revealed that the YesW crystals belong to space group P2(1) and diffract to 2.40 angstrom resolution, with unit-cell parameters a = 56.7, b = 105.6, c = 101.4 angstrom, beta = 94.9 degrees. This is the first report on the crystallization and preliminary X-ray analysis of a family PL-11 rhamnogalacturonan lyase.

DOI： 10.1107/S1744309106011894

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT UNION CRYSTALLOGRAPHY  

Almost all alginate lyases depolymerize alginate in an endolytical fashion via a beta-elimination reaction. The alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, consisting of 776 amino-acid residues, is a novel exotype alginate lyase classified into polysaccharide lyase family 15. The enzyme was crystallized at 293 K by sitting-drop vapour diffusion with polyethylene glycol 4000 as a precipitant. Preliminary X-ray analysis showed that the Atu3025 crystal belonged to space group P2(1) and diffracted to 2.8 angstrom resolution, with unitcell parameters a = 107.7, b = 108.3, c = 149.5 angstrom, beta = 91.5 degrees.

DOI： 10.1107/S1744309106014333

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S166_4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Sphingomonas sp. A1 (strain A1) produces three endotypes (A1-I [65 kDa], A1-II [25 kDa], and A1-III [40 kDa]) and an exotype (A1-IV [86 kDa]) alginate lyases in cytoplasm. These four enzymes cooperatively depolymerize alginate into constituent monosaccharides. In addition to the genes for these lyases, novel genes encoding hypothetical proteins homologous with A1-IV were found in the genomes of many bacteria including strain A1. One such protein, A1-IV'(90 kDa) of strain A1, was overexpressed in Escherichia coli cells, purified, and characterized. A1-IV' catalyzed the cleavage of glycosidic bonds in alginate through a beta-elimination reaction and released unsaturated di- and trisaccharides as main products, thus indicating that the enzyme is an endotype alginate lyase. A1-IV', which differed from A1-IV in some enzymatic properties, was not expressed in strain A1, suggesting that A1-IV' has no significant role in alginate metabolism. A1-IV' and other A1-IV homologs facilitate the creation of novel polysaccharide lyase family 15 based on their primary structures, implying the evolution route of alginate lyases in family PL-15.

DOI： 10.1263/jbb.99.048

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The NAD kinase gene (nadK) of Sphingomonas sp. A1 was cloned and then overexpressed in Escherichia coli, and the gene product (NadK) was purified from the E coli cells through five steps with a 25% yield of activity. NadK was a homodimer of 32 kDa subunits, utilized ATP or other nucleoside triphosphates, but not inorganic polyphosphates, as phosphoryl donors for the phosphorylation of NAD, most efficiently at pH 8.0 and 50-55 degreesC, and was designated as ATP-NAD kinase (NadK). NadK showed no NADH kinase activity and was slightly inhibited by NADP(H). Precursors for NAD biosynthesis such as quinolinic acid, nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, and nicotinic acid had no effect on the NadK activity, as observed in the cases of the NAD kinases of Micrococcus flavus, Mycobacterium tuberculosis, and E coli. Taken together with the report that the NAD kinase of Bacillus subtilis is activated by quinolinic acid [J. Bacteriol. 185 (2003) 4844], it is indicated that the regulatory patterns of NAD kinases differ even among bacterial NAD kinases. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.pep.2004.03.012

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Sphingonionas sp. strain A1 has three endotype alginate lyases (A1-1, A1-II [family PL-7], and A1-III [family PL-5]), each of which is encoded by a single gene. In addition to those of these lyases, a gene (the A1-II' gene) showing significant identity with the A1-II gene was present in the bacterial genome and coded for an alginate lyase with broad substrate specificity. Since no expression of A1-II' was observed even in bacterial cells grown on alginate, the A1-II' gene was thought to be a silent gene derived from the A1-II gene, presumably through duplication, modification, and translocation.

DOI： 10.1128/JB.186.9.2891-2891.2004

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：TAYLOR & FRANCIS LTD  

Food proteins have been identified as a source of bioactive peptides. These peptides are inactive within the sequence of the parent protein and must be released during gastrointestinal digestion, fermentation, or food processing. Of bioactive peptides, multifunctional cationic peptides are more useful than other peptides that have specific activity in promotion of health and/or the treatment of diseases. We have identified and characterized cationic peptides from rice enzymes and proteins that possess multiple functions, including antimicrobial, endotoxin-neutralizing, arginine gingipain-inhibitory, and/or angiogenic activities. In particular, we have elucidated the contribution of cationic amino acids (arginine and lysine) in the peptides to their bioactivities. Further, we have discussed the critical parameters, particularly proteinase preparations and fractionation or purification, in the enzymatic hydrolysis process for producing bioactive peptides from food proteins. Using an ampholyte-free isoelectric focusing (autofocusing) technique as a tool for fractionation, we successfully prepared fractions containing cationic peptides with multiple functions.

DOI： 10.1080/09168451.2016.1277944

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：THE VITAMIN SOCIETY OF JAPAN  

A gram-negative and alginate-assimilating bacterium, Sphingomonas sp. A1, can incorporate the polysaccharide into the cytoplasm through the cell-surface pit and ABC transporter. Alginate is depolymerized into disaccharides to tetrasaccharides by cytoplasmic endotype alginate lyases (A1-I, II, III). An exotype alginate lyase A1-IV degrades the oligosaccharides to unsaturated monosaccharides. α-Keto acids nonenzymatically formed from monosaccharides are converted to 2-keto-3-deoxy-^D-gluconic acid (KDG) by NADPH-dependent reductase A1-R. KDG is eventually metabolized to glyceraldehyde-3-phosphate and pyruvate through the sequential reaction catalyzed by KDG kinase (A1-K) and 2-keto-3-deoxy 6-phosphogluconate aldolase (A1-A). This review deals with the structure and function of bacterial alginate-metabolizing enzymes, especially structure determinants responsible for the catalytic reaction and mode of action of endotype lyases A1-II, II', III in strain A1 and exotype lyase Atu3025 in Agrobacterium tumefaciens as well as for the coenzyme-binding mode of strain A1 α-keto acid reductase (A1-R).

DOI： 10.20632/vso.84.11_525

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