記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MICROBIOL RES FOUNDATION  

Chitinase B from Serratia marcescens 2170 is one of the processive chitinases, and it has a linear path of aromatic amino acid residues on the surface and in the catalytic cleft. There are four surface-exposed residues lined-up towards the cleft, Y481, W479, W252, and Y240. The substitution of these residues with alanine causes a decrease in both the extent of the substrate binding and the hydrolytic activity (Katouno et al., 2004). Here, we examine the three mutants without losing the substrate-binding ability, Y240W, Y481W, and Y240W/Y481W. These mutants were prepared for a detailed analysis of the functions of Y240 and Y481, which showed a lower contribution to substrate binding than W479 and W252. The parameters for the binding of the three mutants to crystalline beta-chitin were similar to those for the wild type. The hydrolytic activity of Y240W and Y240W/Y481W against crystalline beta-chitin was significantly decreased. However, the hydrolytic activity of Y481W was similar to that of the wild type, indicating some differences in the roles of Y240 and Y481 during the processive degradation of crystalline beta-chitin. Taken together with the previous results, it was suggested that while Y240 and Y481 were required for the substrate binding, Y240 had additional roles in the processive degradation of crystalline beta-chitin, possibly in guiding a chitin chain into the catalytic cleft.

DOI： 10.2323/jgam.61.255

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：日本キチン・キトサン学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Processivity refers to the ability of synthesizing, modifying and degrading enzymes to catalyse multiple successive cycles of reaction with polymeric substrates without disengaging from the substrates. Since biomass polysaccharides, such as chitin and cellulose, often form recalcitrant crystalline regions, their degradation is highly dependent on the processivity of degrading enzymes. Here we employ high-speed atomic force microscopy to directly visualize the movement of two processive glycoside hydrolase family 18 chitinases (ChiA and ChiB) from the chitinolytic bacterium Serratia marcescens on crystalline beta-chitin. The half-life of processive movement and the velocity of ChiA are larger than those of ChiB, suggesting that asymmetric subsite architecture determines both the direction and the magnitude of processive degradation of crystalline polysaccharides. The directions of processive movements of ChiA and ChiB are observed to be opposite. The molecular mechanism of the two-way traffic is discussed, including a comparison with the processive cellobiohydrolases of the cellulolytic system.

DOI： 10.1038/ncomms4975

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

A thermally unfolded disulfide-deficient mutant of the starch-binding domain of glucoamylase refolds into a kinetically trapped metastable intermediate when subjected to a rapid lowering of temperature. We attempted to characterise this intermediate using multidimensional NMR spectroscopy. The (1)H-(15)N heteronuclear single quantum coherence spectrum after a rapid temperature decrease (the spectrum of the intermediate) showed good chemical shift dispersion but was significantly different from that of the native state, suggesting that the intermediate adopts a nonnative but well-structured conformation. Large chemical shift changes for the backbone amide protons between the native and the intermediate states were observed for residues in the beta-sheet consisting of strands 2, 3, 5, 6, and 7 as well as in the C-terminal region. These residues were found to be in close proximity to aromatic residues, suggesting that the chemical shift changes are mainly due to ring current shifts caused by the aromatic residues. The two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy experiments showed that the intermediate contained substantial, native-like NOE connectivities, although there were fewer cross peaks in the spectrum of the intermediate compared with that of the native state. It was also shown that there were native-like interresidue NOEs for residues buried in the protein, whereas many of the NOE cross peaks were lost for the residues involved in a surface-exposed aromatic cluster. These results suggest that, in the intermediate, the aromatic cluster at the surface is structurally less organised, whereas the interior of the protein has relatively rigid, native-like side-chain packing. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2011.07.025

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JOHN WILEY & SONS INC  

Refolding of a thermally unfolded disulfide-deficient mutant of the starch-binding domain of glucoamylase was investigated using differential scanning calorimetry, isothermal titration calorimetry, CD, and (1)H NMR. When the protein solution was rapidly cooled from a higher temperature, a kinetic intermediate was formed during refolding. The intermediate was unexpectedly stable compared with typical folding intermediates that have short half-lives. It was shown that this intermediate contained substantial secondary structure and tertiary packing and had the same binding ability with beta-cyclodextrin as the native state, suggesting that the intermediate is highly-ordered and native-like on the whole. These characteristics differ from those of partially folded intermediates such as molten globule states. Far-UV CD spectra showed that the secondary structure was once disrupted during the transition from the intermediate to the native state. These results suggest that the intermediate could be an off-pathway type, possibly a misfolded state, that has to undergo unfolding on its way to the native state.

DOI： 10.1002/pro.188

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

The thermodynamic effects of the disulfide bond of the fragment protein of the starch-binding domain of Aspergillus niger glucoamylase was investigated by measuring the thermal unfolding of the wild-type protein and its two mutant forms, Cys3Gly/Cys98Gly and Cys3Ser/Cys98Ser. The circular dichroism spectra and the thermodynamic parameters of binding with beta-cyclodextrin at 25 degrees C suggested that the native structures of the three proteins are essentially the same. Differential scanning calorimetry of the thermal unfolding of the proteins showed that the unfolding temperature t(1/2) of the two mutant proteins decreased by about 10 degrees C as compared to the wild-type protein at pH 7.0. At t(1/2) of the wild-type protein (52.7 degrees C), the mutant proteins destabilized by about 10 kJ mol(-1) in terms of the Gibbs energy change. It was found that the mutant proteins were quite stabilized in terms of enthalpy, but that a higher entropy change overwhelmed the enthalpic effect, resulting in destabilization.

DOI： 10.1271/bbb.70098

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記述言語：英語   出版者・発行元：社団法人日本農芸化学会  

&nbsp;&nbsp;Thermal unfolding of <i>P. cepacia</i> lipase was observed by adiabatic differential scanning microcalorimetry in the absence and presence of calcium ions at pH 8, and thermodynamic parameters of unfolding were evaluated to analyze the unfolding mechanism of the enzyme. The temperature of unfolding was higher at higher concentrations of Ca²⁺. From the Ca²⁺ concentration-dependence of the unfolding temperature, the number of calcium ions that dissociated from the enzyme molecule upon unfolding was estimated to be one. These results confirmed the validity of the unfolding mechanism proposed previously: NCa²⁺↔D+Ca²⁺, where N and D represent the native and denatured states, respectively, of the enzyme.<br>

DOI： 10.1271/bbb.67.207

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

Serratia marcescens 2170 produces three different types of chitinases and chitin-binding protein CBP21. We found that transposon insertion into the 5 untranslated region (5 UTR) of chiPQ-ctb led to defective chitinase and CBP21 production. ChiX small RNA possessed the complementary sequence of the 5 UTRs of the chiPQ-ctb and chiR and repressed the expression of chiP and chiR. ChiX was detected in a medium containing glucose, glycerol, GlcNAc, and (GlcNAc)(2), but the expression of both chiP and chiR was only observed in a medium containing (GlcNAc)(2). chiX mutant produced chitinases, CBP21, and chitobiase without induction. chiP transcripts were more abundant than those of chiR or chiX in a medium containing (GlcNAc)(2). These results suggest that the constitutively expressed ChiX binds to the highly abundant chiP 5 UTR, thereby leading to the induction of chiR mRNA translation and the subsequent expression of chitinases and CBP21.

DOI： 10.1080/09168451.2015.1083399

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

In order to elucidate the roles of ChiP, ChiQ, and ChiX in chitin utilization by Serratia marcescens 2170, the construction of single-gene deletion mutants of the chiP, chiQ, and chiX genes was attempted by allelic exchange mutagenesis. Delta chiP formed smaller clearing zones and Delta chiX formed larger ones than wild-type 2170 on an agar plate containing colloidal chitin. Delta chiP grew slowly on the lower concentration of (GlcNAc)(2), and there was essentially no growth on chitin oligosaccharides larger than (GlcNAc)(3). The gene product of chiP was detected in the outer membrane fraction, consistently with the hypothesis that chiP encodes outer membrane chitoporin. Deletion of chiQ decreased and that of chiX increased the growth rates on chitin oligosaccharides. These observations strongly suggest that all three genes are involved in chitin utilization and that the deletion mutants obtained in this study might prove useful tools to clarify the details of the chitin utilization system of this bacterium.

DOI： 10.1080/09168451.2014.882755

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MICROBIOL RES FOUNDATION  

The carbon storage regulator (Csr) global regulatory system is conserved in many eubacteria and coordinates the expression of various genes that facilitate adaptation during the major physiological growth phase. The Csr system in Escherichia coli comprises an RNA-binding protein, CsrA; small non-coding RNAs, CsrB and CsrC; and a decay factor for small RNAs, CsrD. In this study, we identified the Csr system in Serratia marcescens 2170. S. marcescens CsrA was 97% identical to E. coli CsrA. CsrB and CsrC RNAs had typical stem-loop structures, including a GGA motif that is the CsrA binding site. CsrD was composed of N-terminal two times transmembrane region and HAMP-like, GGDEF, and EAL domains. Overexpression of S. marcescens csr genes complemented the phenotype of E. coli csr mutants. S. marcescens CsrD affected the decay of CsrB and CsrC RNAs in E. coli. These results suggest that the Csr system in S. marcescens is composed of an RNA-binding protein, two Csr small RNAs, and a decay factor for Csr small RNAs.

DOI： 10.2323/jgam.60.79

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

Chitinase A1 (ChiA1) from Bacillus circulans WL-12 comprises an N-terminal catalytic domain, two fibronectin type III domains, and a C-terminal chitin-binding domain (ChBD). The ChBD of ChiA1 (ChBD(ChiA1)) belongs to carbohydrate-binding module (CBM) family 12 and specifically binds to insoluble or crystalline chitin. It has been suggested that tryptophan-687 (Trp687) is involved in the chitin-binding activity of this ChBD. Site-directed mutagenesis was used to identify additional amino acid residues required for chitin-binding activity of this domain. Furthermore, a total of 14 amino acid residues in ChBD(ChiA1) were carefully selected, and it was found that mutation of Gln679, which is not well-conserved in CBM family 12, significantly decreased the binding activity to colloidal chitin. A nuclear magnetic resonance study demonstrated that neither the Q679A nor the W687A mutation altered the overall structure of ChBD(ChiA1). Therefore, Gln679 was identified as a new residue that is involved in the chitin-binding activity of ChBD(ChiA1) in addition to Trp687. However, the mechanism of chitin binding by ChBD is still unknown.

DOI： 10.1093/jb/mvt043

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

Serratia marcescens 2170 produces three chitinases and the chitin-binding protein CBP21, and efficiently degrades insoluble and crystalline chitin. The three chitinases and CBP21 are induced by N,N'-diacetylchitobiose [(GlcNAc)(2)], the major product of chitin hydrolysis by S. marcescens chitinases. We have found that uptake of both (GlcNAc)(2) and N-acetylglucosamine (GlcNAc) is important for the efficient utilization of (GlcNAc)(2) because (GlcNAc)(2) is less efficiently fermented by S. marcescens 2170 in the absence of chitobiase. In order to determine the mechanism of utilization of the degradation products of chitin by S. marcescens, chitobiase deficient transposon mutants were screened. A transposon present in chitobiase-deficient mutants was inserted into the ybfMN-ctb cluster. The mutants produced chitinases, except for TT327, in which a transposon was inserted into the 5'-untranslated region (5'-UTR) of ybfM. Ectopic expression of this region in TT327 restored chitinase production. These results indicate that the 5'-UTR of ybfM is important for chitinase induction in S. marcescens.

DOI： 10.1271/bbb.120403

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

We isolated two major zwitterionic glycosphingolipids (ZGLs) from the phytopathogenic filamentous fungus Trichoderma viride. Structural analyses showed that the ZGLs (designated Tv-ZGL2 and Tv-ZGL3) were the same as the glycosphingolipids ZGL2 and ZGL4 from Acremonium sp., which are described in our previous paper. ZGLs have the following structure: Man(alpha 1-6)GlcN(alpha 1-2)Ins-P-Cer (Tv-ZGL2) and phosphocholine (PC) -&gt; 6Man(alpha 1-6)GlcN(alpha 1-2)Ins-P-Cer (Tv-ZGL3). To determine whether these ZGLs have functional roles in plant-fungus interaction, we tested to determine whether they would induce defense responses in cultured rice cells. We found that T. viride's ZGLs elicited expression of the PAL and PBZ1 genes, both of which are associated with pathogen resistance. Tv-ZGL2 induced cell death at a moderate rate. Tv-ZGL3, which contains a PC moiety, induced a high level of cell death in rice cells.

DOI： 10.1271/bbb.80480

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Recently, a gene cluster involving a phosphorylase specific for lacto-N-biose I (LNB; Gal beta 1-3GlcNAc) and galacto-N-biose (GNB; Gal beta 1-3GalNAc) has been found in Bifidobacterium longum. We showed that the solute-binding protein of a putative ATP-binding cassette-type transporter encoded in the cluster crystallizes only in the presence of LNB or GNB, and therefore we named it GNB/LNB-binding protein (GL-BP). Isothermal titration calorimetry measurements revealed that GL-BP specifically binds LNB and GNB with K-d values of 0.087 and 0.010 mu M, respectively, and the binding process is enthalpy-driven. The crystal structures of GL-BP complexed with LNB, GNB, and lacto-N-tetraose ( Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc) were determined. The interactions between GL-BP and the disaccharide ligands mainly occurred through water-mediated hydrogen bonds. In comparison with the LNB complex, one additional hydrogen bond was found in the GNB complex. These structural characteristics of ligand binding are in agreement with the thermodynamic properties. The overall structure of GL-BP was similar to that of maltose-binding protein; however, the mode of ligand binding and the thermodynamic properties of these proteins were significantly different.

DOI： 10.1074/jbc.M709777200

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記述言語：英語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.48.S87_3

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