担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Thousands of terpenes have been identified to date. However, only two classes of enzymes are known to be involved in their biosynthesis, and each class has characteristic amino-acid motifs. We recently identified a novel large-terpene (C25/C30/C35) synthase, which shares no motifs with known enzymes. To elucidate the molecular mechanism of this enzyme, we determined the crystal structure of a large-β-prene synthase from B. alcalophilus (BalTS). Surprisingly, the overall structure of BalTS is similar to that of the α-domain of class I terpene synthases although their primary structures are totally different from each other. Two novel aspartate-rich motifs, DYLDNLxD and DY(F,L,W)IDxxED, are identified, and mutations of any one of the aspartates eliminate its enzymatic activity. The present work leads us to propose a new subclass of terpene synthases, class IB, which is probably responsible for large-terpene biosynthesis.

DOI： 10.1039/c8sc00289d

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担当区分：責任著者   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 CHEMICAL SOC  

The onoceroids are triterpenoids biosynthesized from squalene or (3S)-2,3-oxidosqualene by cyclization from both termini. We recently revealed that tetraprenyl-beta-curcumene cyclase from Bacillus megaterium (BmeTC) is a bifunctional triterpene/sesquarterpene cyclase that converts head-to-tail tetraprenyl-beta-curcumene and tail-to-tail squalene into pentacyclic and bicyclic products, respectively, in vivo. Here, we reveal that BmeTC has an unprecedented catalytic function in cyclizing squalene from both termini and is the first onoceroid synthase. We also report the first onoceroids from bacterial origin. Our discoveries suggest that symmetric and asymmetric onoceroids could be biosynthesized by a single enzyme via an intermediate cyclized at one terminus of squalene. Furthermore, the new function of BmeTC enabled the synthesis of (+)-ambrein, a major constituent of ambergris that is difficult to obtain naturally, via a mutated squalene-hopene cyclase-catalyzed reaction from easily available squalene.

DOI： 10.1021/ja4107226

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/ja2060319

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/ja203779h

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Onoceroids are a rare family of triterpenes. One representative onoceroid is ambrein, which is the main component of ambergris used as a traditional medicine. We have previously identified the onoceroid synthase, BmeTC, in Bacillus megaterium and succeeded in creating ambrein synthase by introducing mutations into BmeTC. Owing to the structural similarity of ambrein to vitamin D, a molecule with diverse biological activities, we hypothesized that some of the activities of ambergris may be induced by the binding of ambrein to the vitamin D receptor (VDR). We demonstrated the VDR binding ability of ambrein. By comparing the structure-activity relationships of triterpenes with both the VDR affinity and osteoclastic differentiation-promoting activity, we observed that the activity of ambrein was not induced via the VDR. Therefore, some of the activities of ambergris, but not all, can be attributed to its VDR interaction. Additionally, six unnatural onoceroids were synthesized using the BmeTC reactions, and these compounds exhibited higher VDR affinity than that of ambrein. Enzymatic syntheses of onoceroid libraries will be valuable in creating a variety of bioactive compounds beyond ambergris.

DOI： 10.1038/s41598-024-52013-7

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掲載種別：研究論文（学術雑誌）   出版者・発行元：The Chemical Society of Japan  

DOI： 10.1246/cl.230151

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Terpene synthase (TS) from Bacillus alcalophilus (BalTS) is the only Class IB TS for which a 3D structure has been elucidated. Recently, geranyl-β-phellandrene, a novel cyclic diterpene, was identified as a product of BalTS in addition to the acyclic β-springene. In the present study, we have provided insight into the mechanism of geranyl-β-phellandrene formation. Deuterium labeling experiments revealed that the compound is produced via a 1,3-hydride shift. In addition, nonenzymatic reactions using divalent metal ions were performed. The enzyme is essential for the geranyl-β-phellandrene formation. Furthermore, BalTS variants targeting tyrosine residues enhanced the yield of geranyl-β-phellandrene and the proportion of the compound of the total products. It was suggested that the expansion of the active site space may allow the conformation of the intermediates necessary for cyclization. The present study describes the first Class IB TSs to successfully alter product profiles while retaining high enzyme activity.

DOI： 10.1093/bbb/zbac036

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Delamanid (DLM), a nitro-dihydroimidazooxazole derivative currently approved for pulmonary multidrug-resistant tuberculosis (TB) therapy, is a prodrug activated by mycobacterial 7,8-didemethyl-8-hydroxy 5-deazaflavin electron transfer coenzyme (F420)-dependent nitroreductase (Ddn). Despite inhibiting the biosynthesis of a subclass of mycolic acids, the active DLM metabolite remained unknown. Comparative liquid chromatography-mass spectrometry (LC-MS) analysis of DLM metabolites revealed covalent binding of reduced DLM with a nicotinamide ring of NAD derivatives (oxidized form) in DLM-treated Mycobacterium tuberculosis var. Bacille de Calmette et Guérin. Isoniazid-resistant mutations in the type II NADH dehydrogenase gene (ndh) showed a higher intracellular NADH/NAD ratio and cross-resistance to DLM, which were restored by complementation of the mutants with wild-type ndh Our data demonstrated for the first time the adduct formation of reduced DLM with NAD in mycobacterial cells and its importance in the action of DLM.

DOI： 10.1128/AAC.01755-19

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

Delamanid (DLM), a nitro-dihydroimidazooxazole derivative currently approved for pulmonary multidrug-resistant tuberculosis (TB) therapy, is a prodrug activated by mycobacterial 7,8-didemethyl-8-hydroxy 5-deazaflavin electron transfer coenzyme (F
₄₂₀
)-dependent nitroreductase (Ddn). Despite inhibiting the biosynthesis of a subclass of mycolic acids, the active DLM metabolite remained unknown. Comparative liquid chromatography-mass spectrometry (LC-MS) analysis of DLM metabolites revealed covalent binding of reduced DLM with a nicotinamide ring of NAD derivatives (oxidized form) in DLM-treated
<named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
var.

DOI： 10.1128/aac.01755-19

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

© 2020 The Author(s) Class I terpene synthase (TPS) generates bioactive terpenoids with diverse backbones. Sesterterpene synthase (sester-TPS, C25), a branch of class I TPSs, was recently identified in Brassicaceae. However, the catalytic mechanisms of sester-TPSs are not fully understood. Here, we first identified three nonclustered functional sester-TPSs (AtTPS06, AtTPS22, and AtTPS29) in Arabidopsis thaliana. AtTPS06 utilizes a type-B cyclization mechanism, whereas most other sester-TPSs produce various sesterterpene backbones via a type-A cyclization mechanism. We then determined the crystal structure of the AtTPS18–FSPP complex to explore the cyclization mechanism of plant sester-TPSs. We used structural comparisons and site-directed mutagenesis to further elucidate the mechanism: (1) mainly due to the outward shift of helix G, plant sester-TPSs have a larger catalytic pocket than do mono-, sesqui-, and di-TPSs to accommodate GFPP; (2) type-A sester-TPSs have more aromatic residues (five or six) in their catalytic pocket than classic TPSs (two or three), which also determines whether the type-A or type-B cyclization mechanism is active; and (3) the other residues responsible for product fidelity are determined by interconversion of AtTPS18 and its close homologs. Altogether, this study improves our understanding of the catalytic mechanism of plant sester-TPS, which ultimately enables the rational engineering of sesterterpenoids for future applications.

DOI： 10.1016/j.xplc.2020.100051

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出版者・発行元：Oxford University Press (OUP)  

<jats:title>ABSTRACT</jats:title>
<jats:p>We determined if Japanese Rice Wine (Sake) had inhibitory effects on stress-induced enhancement of masseter muscle (MM) nociception in the rats. Male rats were subjected to the repeated forced swim stress (FS) or sham conditionings from Day −3 to −1. Daily administration of Sake or saline was conducted after each stress conditioning. At Day 0 the number of Fos positive cells, a marker for neural activity, was quantified at the trigeminal subnucleus caudalis (Vc) region by MM injury with formalin. FS increased MM-evoked Fos expression in the Vc region, which was inhibited by Sake compared to saline administration. Sake did not alter the number of Fos positive cells under sham conditions, indicating that inhibitory roles of Sake on neural activity in the Vc region were seen under FS conditions. These findings indicated that Sake had inhibitory roles on stress-induced MM nociception at the Vc region in our experimental conditions.</jats:p>

DOI： 10.1080/09168451.2018.1524705

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その他リンク： https://search.jamas.or.jp/link/ui/2019253806

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Rhododendron dauricum produces daurichromenic acid, an anti-HIV meroterpenoid, via oxidative cyclization of the farnesyl group of grifolic acid. The prenyltransferase (PT) that synthesizes grifolic acid is a farnesyltransferase in plant specialized metabolism. In this study, we demonstrated that the isoprenoid moiety of grifolic acid is derived from the 2-C-methyl-d-erythritol-4-phosphate pathway that takes place in plastids. We explored candidate sequences of plastid-localized PT homologs and identified a cDNA for this PT, RdPT1, which shares moderate sequence similarity with known aromatic PTs. RdPT1 is expressed exclusively in the glandular scales, where daurichromenic acid accumulates. In addition, the gene product was targeted to plastids in plant cells. The recombinant RdPT1 regiospecifically synthesized grifolic acid from orsellinic acid and farnesyl diphosphate, demonstrating that RdPT1 is the farnesyltransferase involved in daurichromenic acid biosynthesis. This enzyme strictly preferred orsellinic acid as a prenyl acceptor, whereas it had a relaxed specificity for prenyl donor structures, also accepting geranyl and geranylgeranyl diphosphates with modest efficiency to synthesize prenyl chain analogs of grifolic acid. Such a broad specificity is a unique catalytic feature of RdPT1 that is not shared among secondary metabolic aromatic PTs in plants. We discuss the unusual substrate preference of RdPT1 using a molecular modeling approach. The biochemical properties as well as the localization of RdPT1 suggest that this enzyme produces meroterpenoids in glandular scales cooperatively with previously identified daurichromenic acid synthase, probably for chemical defense on the surface of R. dauricum plants.

DOI： 10.1104/pp.18.00655

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The acyclic molecule squalene (1) is cyclized into 6,6,6,6,5-fused pentacyclic hopene (2) and hopanol (3; ca. 5:1) through the action of Alicyclobacillus acidocaldarius squalene-hopene cyclase (AaSHC). The polycyclization reaction proceeds with regio- and stereochemical specificity under precise enzymatic control. This pentacyclic hopane skeleton is generated by folding 1 into an all-chair conformation. The Ala306 residue in AaSHC is conserved in known squalene-hopene cyclases (SHCs); however, increasing the steric bulk (A306T and A306V) led to the accumulation of 6,6,6,5-fused tetracyclic scaffolds possessing 20R stereochemistry in high yield (94 % for A306V). The production of the 20R configuration indicated that 1 had been folded in a chair-chair-chair-boat conformation; in contrast, the normal chair-chair-chair-chair conformation affords the tetracycle with 20S stereochemistry, but the yield produced by the A306V mutant was very low (6 %). Consequently, bulk at position 306 significantly affects the stereochemical fate during the polycyclization reaction. The SHC also accepts (3R) and (3S)-2,3-oxidosqualenes (OXSQs) to generate 3α,β-hydroxyhopenes and 3α,β-hydroxyhopanols through polycyclization initiated at the epoxide ring. However, the Val and Thr mutants generated epoxydammarane scaffolds from (3R)-OXSQ; this indicated that the polycyclization cascade started in these instances at the terminal double bond position. This work is the first to report the polycyclization of oxidosqualene starting at the terminal double bond.

DOI： 10.1002/cbic.201800281

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROYAL SOC CHEMISTRY  

The first chemical synthesis of pentacyclic onocerane triterpenoids has been achieved. A putative biomimetic tricyclization cascade is employed to forge a fused decalin-/oxepane ring system. The synthetic route proceeds to (+)-cupacinoxepin in seven steps and to (+)-onoceranoxide in eight steps in the longest linear sequence, when starting from geranyl chloride and (+)-sclareolide. The bioinspired epoxypolyene cyclization is supported by computational and enzymatic studies.

DOI： 10.1039/c7sc03903d

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-V C H VERLAG GMBH  

Onoceroids are a group of triterpenes biosynthesized from squalene or dioxidosqualene by cyclization from both termini. We previously identified a bifunctional triterpene/sesquarterpene cyclase (TC) that constructs a tetracyclic scaffold from tetraprenyl--curcumene (C-35) but a bicyclic scaffold from squalene (C-30) in the first reaction. TC also accepts the bicyclic intermediate as a substrate and generates tetracyclic and pentacyclic onoceroids in the second reaction. In this study, we analyzed the catalytic mechanism of an onoceroid synthase by using mutated enzymes. TCY167A produced an unnatural tricyclic triterpenol, but TCY167L, TCY167F, and TCY167W formed small quantities of tricyclic compounds, which suggested that the bulk size at Y167 contributed to termination of the cyclization of squalene at the bicyclic step. Our findings provide insight into the unique catalytic mechanism of TC, which triggers different cyclization modes depending on the substrate. These findings may facilitate the large-scale production of an onoceroid for which natural sources are limited.

DOI： 10.1002/cbic.201700329

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-V C H VERLAG GMBH  

We performed functional analysis of recombinant enzymes and analysis of isoprenoid metabolites in Bacillus clausii to gain insights into the biosynthesis of rare terpenoid groups of sesterterpenes, head-to-tail triterpenes, and sesquarterpenes. We have identified an (all-E)-isoprenyl diphosphate synthase (E-IDS) homologue as a trifunctional geranylfarnesyl diphosphate (GFPP)/hexaprenyl diphosphate (HexPP)/heptaprenyl diphosphate (HepPP) synthase. In addition, we have redefined the function of a tetraprenyl--curcumene synthase homologue as that of a trifunctional sesterterpene/triterpene/sesquarterpene synthase. This study has revealed that GFPP, HexPP, and HepPP, intermediates of two isoprenoid pathways (acyclic terpenes and menaquinones), are biosynthesized by one trifunctional E-IDS. In addition, GFPP/HexPP and HepPP are the primary substrates for the biosynthesis of acyclic terpenes and menaquinone-7, respectively.

DOI： 10.1002/cbic.201500138

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

Little is known of the biosynthesis of sesquarterpenes and the synthesis of unnatural terpenoids by sesquarterpene biosynthetic enzymes has not yet been reported. In this study, the enzymatic cyclization of head-to-tail acyclic triterpene beta-hexaprene a natural product isolated from Bacillus clausii-using tetraprenyl-beta-curcumene cyclase (TC) from Bacillus subtilis resulted in the formation of two unnatural pentacyclic triterpenes. It was revealed that B. subtilis TC, which forms tetracyclic terpenoid scaffold from tetraprenyl-beta-curcumene in vivo, could be used to construct the 6/6/6/6/6-fused pentacyclic scaffold in vitro, suggesting that the active site cavity of TC has sufficient space to accommodate this unnatural pentacyclic scaffold. This is the first report demonstrating the utility of a sesquarterpene cyclase toward the synthesis of unnatural terpenoids. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tetlet.2013.09.135

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-V C H VERLAG GMBH  

DOI： 10.1002/cbic.201300035

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

A new sesquarterpene, heptaprenylcycli-14E,18E-diene, was isolated from Mycobacterium chlorophenolicum cells grown up to the stationary phase. The absence of heptaprenylcycli-14Z,18E-diene indicates that only (E,E,E)-geranylgeranyl diphosphate may be utilized as an intermediate of sesquarterpene biosynthesis in the stationary phase, in contrast with the logarithmic growth phase in which both (E,E)-farnesyl diphosphate and (E,E,E)-geranylgeranyl diphosphate are used. Further, our findings suggest that the step-wise reduction of the polyprenyl group in sesquarterpene biosynthesis might proceed in a different order from that in chlorophyll biosynthesis. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tetlet.2012.03.019

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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 CHEMICAL SOC  

The gene products of AK121211, AK066327, and AK070534 from Oryza sativa encode cycloartenol, parkeol, and achilleol B synthases, respectively. Parkeol synthase is a unique enzyme that affords parkeol as a single product. Achilleol B synthase is the third seco-type triterpene cyclase identified to date, and triterpenes produced by this synthase include achilleol B (90%), tetracyclic (5.12%) and pentacyclic scaffolds (4.37%), and unidentified triterpenes (0.51%). The pathway for achilleol B biosynthesis is proposed.

DOI： 10.1021/ol200777d

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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 Rv3377c and Rv3378c genes from Mycobacterium tuberculosis are specifically found in the virulent Mycobacterium species, but not in the avirulent species. The Rv3378c-encoded enzyme produced tuberculosinol 2 (5(6), 13(14)-halimadiene-15-ol), 13R-5a and 13S-isotuberculosinol 5b (5(6), 14(15)-halimadiene-13-ol) as its enzymatic products from tuberculosinyl diphosphate 3, indicating that the Rv3378c enzyme catalyzed the nucleophilic addition of a water molecule after the release of a diphosphate moiety. The three enzymatic products 2, 5a, and 5b were produced irrespective of the N- and C-terminal His-tagged Rv3378c enzymes, and of the maltose-binding protein fusion enzyme; the product distribution ratio was identical between the enzymes as 1:1 for 2:5, and 1:3 for 5a:5b. The successful separation of 5a and 5b by a chiral HPLC column provided the first complete assignments of H-1- and C-13-NMR data for 5a and 5b. The enzymatic mechanism for producing 2, 5a, and 5b is proposed here, and the optimal catalytic conditions and kinetic parameters, in addition to the divalent metal effects, are described. Site-directed mutagenesis of Asp into Asn, targeted at the DDXXD motif, resulted in significantly decreased enzymatic activity.

DOI： 10.1271/bbb.100570

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

Nonpathogenic Mycobacterium species produce rare cyclic C-35 terpenes that are biosynthesized by cyclization of Z-type C-35 polyprenyl diphosphate. To provide deeper insight into the biosynthesis of C-35 terpenes, we carried out functional analyses of three Z-prenyltransferase homologues in M. vanbaalenii identified by genomic analysis. Mvan_3822, a novel bifunctional Z-prenyltransferase, biosynthesizes C-35-heptaprenyl diphosphate as a main product from (E,E)-farnesyl diphosphate (E,E-FPP) and (E,E,E)-geranylgeranyl diphosphate (E,E,E-GGPP), but produces a C-50-decaprenyl diphosphate from geranyl diphosphate. Mvan_1705 is a novel Z,E,E-GGPP synthase. In addition, novel cyclic C-35 terpenes, (14E)- and (14Z)-dehydroheptaprenylcycline, were identified as minor metabolites in nonpathogenic Mycobacterium cells. C-35 terpenes could be biosynthesized by two routes, in which E and Z geometric isomers of heptaprenyl diphosphate are produced from E,E-FPP and E,E,E-GGPP, and the prenylreductase responsible for the biosynthesis of C-35 terpenes could reduce both E and Z prenyl residues.

DOI： 10.1002/cbic.201000328

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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：PERGAMON-ELSEVIER SCIENCE LTD  

The enzymatic reaction of copalyl diphosphate (CDP), ent-CDP, syn-CDP, and tuberculosinyl diphosphate with the CYC2 enzyme from Kitasatospora griseola afforded sclarene, biformene, and novel diterpenes having a buta-1,3-diene moiety in the side chain. The substrate specificity of the CYC2 is discussed. (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tetlet.2009.10.110

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担当区分：筆頭著者,　責任著者   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  

A search for C-35-terpenes from non-saponified extracts of 12 non-pathogenic Mycobacterium species was carried out. Octahydroheptaprenyl mycolic acyl esters were isolated from M. chlorophenolicum cells which were also found from M. thermoresistibile, M. vanbaalenii, M. aichiense, M. smegmatis, and M. parafortuitum. This is the first report on a polyprenol esterified by a mycolate. A novel monocyclic C-35-terpene possessing a ketone, named heptaprenylcycline B, was isolated, which was detected from M. chlorophenolicum and M. vanbaalenii. The biosynthetic pathway to heptaprenylcycline B was investigated with ancymidol which acts as an inhibitor of a P450 monooxygenase. This experiment suggested that the P450 monooxygenase may be responsible for the production of heptaprenylcycline B.

DOI： 10.1271/bbb.90669

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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：WILEY-V C H VERLAG GMBH  

Squalene-hopene cyclase (SHC) catalyzes the polycyclization of squalene (C-30) to the pentacyclic hopene With regio- and stereochemical specificity. In this study, we reviewed the polycyclization reaction of the C-35 polyprenoid catalyzed by SHC. Surprisingly, our results completely disagreed with a previous publication in which it was reported that a hexacyclic skeleton was constructed as the single product in 10% yield (I. Abe. H. Tanaka, H. Noguchi, J. Am. Chem. Soc. 2002, 124, 14514-14515). Our experimental results showed that many tri- and tetracyclic products, up to 12. including novel carbocyclic cores, were generated in a high conversion ratio (97%), but no detectable amounts of the penta- and hexacycle were produced. The mechanisms for the formation of the C-31 Polyprene products isolated by us are discussed in this paper. The following four conformations were generated during the polycyclization cascade: chair-chair-boat, chair-chair-chair, chairchair-chair-boat, and chair-chair-chair-chair. Larger amounts of the false intermediates with 13 alpha-H (tricycle) and 17 alpha-H (tetracycle) were produced compared with the true intermediates (13 beta-H and 17 beta-H), which indicates that the C-35 polyprene cannot fold correctly in the enzyme cavity due to the extra C-5 unit appended to squalene. This Would have promoted the formation of the aborted cyclization products With tri- and tetracycles. In addition, the fact that no penta- or hexacyclic products were formed further indicates that SHC does not have sufficient space to accommodate the entire carbon framework of C-31.

DOI： 10.1002/chem.200802142

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROYAL SOC CHEMISTRY  

Lipid components from 12 nonpathogenic Mycobacterium species were analysed. A novel cyclic C35-terpene, named heptaprenylcycline 1, was obtained from 3 species, while octahydroheptaprenol 2, which has 3 Z-double bonds, was obtained from 6 species. The amounts of 1 and 2 in the cultured cells increased after the 4- to 6-d stationary phase. The yield of I was considerably greater at a higher temperature of 37 degrees C than at an optimal temperature of 28 degrees C, while that of 2 remained unchanged at all temperatures. A feeding experiment with D-[1(-13) C]glucose revealed that 1 was produced via isopentenyl diphosphate, which is a metabolite of glycolysis and the methylerythritol phosphate pathway. The conversion of octahydroheptaprenyl diphosphate 2-PP to 1 was successful by using the cell-free extracts of M. chlorophenolicum, demonstrating that 2-PP is the biosynthetic intermediate of 1. This is the first example of the biosynthesis of a natural terpene via the cyclisation of a linear C-35-isoprenoid. The substrate 2-PP for C-35-terpene cyclase has Z-type prenyl moieties; however, terpene cyclases usually employ E-type isoprenoids. The gene encoding the terpene cyclase that cyclises prenyl diphosphate containing Z-double bonds as the natural substrate has not yet been detected. Despite a careful search using the FASTA3 program, we could not detect any gene that is homologous to the known diphosphate-triggered type of mono-, sesqui- and diterpene cyclases in the genome of M. vanbaalenii, the DNA sequence of which has recently been elucidated. This suggests that a novel type of terpene cyclase might exist in the nonpathogenic Mycobacterium species.

DOI： 10.1039/b808513g

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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  

Sterol biosynthesis by prokaryotic organisms is very rare. Squalene epoxidase and lanosterol synthase are prerequisite to cyclic sterol biosynthesis. These two enzymes, from the methanotrophic bacterium Methylococcus capsulatus, were functionally expressed in Escherichia coli. Structural analyses of the enzymatic products indicated that the reactions proceeded in a complete regio- and stereospecific fashion to afford (3S)2,3-oxidosqualene from squalene and lanosterol from (3S)-2,3-oxidosqualene, in full accordance with those of eukaryotes. However, our result obtained with the putative lanosterol synthase was inconsistent with a previous report that the prokaryote accepts both (3R)and (3S)-2,3-oxidosqualenes to afford 3-epi-lanosterol and lanosterol, respectively. This is the first report demonstrating the existence of the genes encoding squalene epoxidase and lanosterol synthase in prokaryotes by establishing the enzyme activities. The evolutionary aspect of prokaryotic squalene epoxidase and lanosterol synthase is discussed.

DOI： 10.1271/bbb.70331

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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：ROYAL SOC CHEMISTRY  

A biosynthetic intermediate of violacein produced by the mixed enzymes of VioABDE was elucidated to be 5-( 5- hydroxy1H- indol- 3- yl)- 3-( 1H- indol- 3- yl)- 1H- pyrrole- 2- carboxylic acid, named protoviolaceinic acid, indicating that VioC, responsible for the final biosynthetic step, works to oxygenate at the 2- position of the right side indole ring, and that the oxygenation reaction to form the central pyrrolidone core proceeds in a nonenzymatic fashion.

DOI： 10.1039/b705358d

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記述言語：英語   出版者・発行元：天然有機化合物討論会  

DOI： 10.24496/intnaturalprod.2006.0__P-295_

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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：ROYAL SOC CHEMISTRY  

The cloning and functional expression of Mycobacterium tuberculosis Rv3377c in Escherichia coli revealed that this gene encodes the diterpene cyclase for producing (+)-5(6),13-halimadiene-15-ol, which accepts geranylgeranyldiphosphate as the intrinsic substrate.

DOI： 10.1039/b415346d

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担当区分：筆頭著者   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  

To provide insight into the catalytic mechanism for the final deprotonation reaction of squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius, mutagenesis experiments were conducted for the following ten residues: Thr41, Glu45, Glu93, Arg127, Trp133, Gln262, Pro263, Tyr267, Phe434 and Phe437. An X-ray analysis of SHC has revealed that two types of water molecules ("front water" and "back waters") were involved around the deprotonation site. The results of these mutagenesis experiments allow us to propose the functions of these residues. The two, residues of Gln262 and Pro263 probably work to keep away the isopropyl group of the hopanyl cation intermediate from the "front water molecule," that is, to place the "front water" in a favorable position, leading to the minimal production of by-products, i.e., hopanol and hop-21(22)-ene. The five residues of Thr41, Glu45, Glu93, Arg127 and Trp133, by which the hydrogen-bonded network incorporating the "back waters" is constructed, increase the polarization of the "front water" to facilitate proton elimination from the isopropyl moiety of the hopanyl cation, leading to the normal product, hop22(29)-ene. The three aromatic residues of Tyr267, Phe434 and Phe437 are likely to play an important role in guiding squalene from the enzyme surface to the reaction cavity (substrate channeling) by the strong affinity of their aromatic residues to the squalene substrate.

DOI： 10.1271/bbb.68.728

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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：ROYAL SOC CHEMISTRY  

To provide insight into the polycyclization mechanism of squalene by squalene-hopene cyclase (SHC) from Alicyclobacilus acidocaldarius, some analogs of nor- and bisnorsqualenes were synthesized including the deuterium-labeled squalenes and incubated with the wild-type SHC, leading to the following inferences. (1) The deprotonation reaction for the introduction of the double bond of the hopene skeleton occurs exclusively from the Z-methyl group on the terminal double bond of squalene. (2) 3R-Oxidosqualene was folded in a boat conformation for the A-ring construction, while the 3S-form was in a chair structure. (3) The terminal two methyl groups are indispensable both for the formation of the 5-membered E-ring of the hopene skeleton and for the initiation of the polycyclization cascade, but the terminal Z-methyl group has a more crucial role for the construction of the 5-membered E-ring than the E-methyl group. (4) Some of the novel terpene skeletons, 36, 37, 39 and 40, were created from the analogs employed in this investigation.

DOI： 10.1039/b401172d

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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：WILEY-V C H VERLAG GMBH  

DOI： 10.1002/anie.200461523

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担当区分：筆頭著者   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 functions of Tyr420 and Leu607 were analyzed by constructing various site-directed mutants. The mutation at position 420 into Ala and Gly gave bicyclic alpha-and gamma-polypodatetraene in significant amounts, but with a trace amount of tricyclic malabaricatriene. The kinetic data for and the product distribution of the Y420F mutant indicate that the major function of Tyr420 is to stabilize the 6/6-fused bicyclic cation. Mutation experiments on Leu607 demonstrate that the appropriate steric bulk size at position 607 is required to strongly bind with the product-like conformation formed during the polycyclization process. Introduction of the bulkiest Trp residue into 420 or 607 led to the production of a novel monocyclic triterpene having the (5R,6R)-1,5,6-trimethylcyclohexene ring, named neoachillapentaene, indicating that the enzymatic cyclization proceeded via a constrained boat structure. Folding of the squalene molecule into a boat conformation by squalene cyclase has not been reported before.

DOI： 10.1271/bbb.66.1660

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担当区分：筆頭著者   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  

Site-directed mutagenesis experiments on all the conserved residues of Phe and Tyr in all the known squalene-hopene cyclases (SHCs) were carried out to identify the active site residues of thermophilic Alicyclobacillus acidocaldarius SHC. The following functions are proposed on the basis of kinetic data and trapping of the prematurely cyclized products: (1) The Y495 residue probably amplifies the D376 acidity, which is assumed to work as a proton donor for initiating the polycyclization cascade, but its role is moderate. (2) Y609 possibly assists the function of F365, which has previously been assigned to exclusively stabilize the C-8 carbocation intermediate through cation-pi interaction. The Y609A mutant produced a partially cyclized bicyclic triterpene. (3) Y612 works to stabilize both the C10 and C8 carbocations, this being verified by the finding that mono- and bicyclic products were formed with the Y612A mutant. (4) F129 was first identified to play a crucial role in catalysis. (5) The three residues, Y372, V474 and Y540, are responsible for reinforcing the protein structure against thermal denaturation, Y474 being located inside QW motif 3.

DOI： 10.1271/bbb.65.2233

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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：ROYAL SOC CHEMISTRY  

Incubation of squalene with the site-directed mutant F605A of squalene-hopene cyclase from Alicyclobacillus acidocaldarius yielded many triterpenes consisting of the 6/6/5-fused tri-, 6/6/6/5-fused tetra-, and 6/6/6/6/5-fused pentacyclic skeletons, the function of F605 being assignable for facilitating the ring expansion and for stabilizing the hopanyl C22-cation, possibly via cation-pi interactions.

DOI： 10.1039/b004129g

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担当区分：筆頭著者   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 clarify the function of the DXDDTA motif in squalene-hopene cyclase and to identify the acidic amino acid residues crucial for the catalysis, site-directed mutagenesis experiments were carried out. The following results were found: (1) residues D374 and D376 work for the initiation of polyolefin cyclization which arises from the proton attack on the terminal double bond; (2) residue D377 stabilizes C-10 carbocation of the initially cyclized A-ring intermediate, leading to subsequent B-ring closure, which was further verified by isolating the partially cyclized monocyclic product; (3) residues D313 and D447 outside the DXDDTA motif were identified as new active sites; (4)the H451 residue is likely to work in the protonated form to enhance the acidity of the carboxyl groups of D374 and/or D376.

DOI： 10.1271/bbb.63.2189

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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：ROYAL SOC CHEMISTRY  

Two bicyclic products were accumulated by the mutant F365A, showing the amino acid residue is located close to the transient C-8 carbocation intermediate in the active site cavity; the mutants of F365Y and F365W significantly accelerated the cyclization reaction at low temperatures.

DOI： 10.1039/a905559b

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担当区分：筆頭著者   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  

Site-directed mutagenesis experiments were carried out to identify the responsibility of the eight QW motifs for the reaction catalyzed by squalene-hopene cyclase (SHC). Alterations of the conserved tryptophans, which are responsible for the stacking structure with glutamine, into aliphatic amino acids gave a significantly lower temperature for the catalytic optimum as for the mutageneses of QW motifs 4, 5a and 5b, which are specifically present in SHCs. However, there was no change in the optimal temperatures of the mutated SHCs targeted at the other five motifs 1, 2, 3, 5c and 6. Thus, reinforcement against heat denaturation can be proposed as a function of the three QW motifs 4, 5a and 5b, but no function could be identified for the QW motifs 1, 2, 3, 5c and 6, although they are commonly found in all the families of prokaryotic SHCs and eukaryotic oxidosqualene cyclases. On the other hand, the three conserved tryptophans of W169, W312 and W489, which are located inside the putative central cavity and outside the QW motifs, were identified as components of the active sites, but also had a function against thermal denaturation. The other two tryptophan residues of W142 and W558, which are located outside the QW motifs, were found not to be active sites, but also had a role for stabilizing the protein structure. It is noteworthy that the mutants replaced by phenylalanine had higher temperatures for the catalytic optimum than those replaced by aliphatic amino acids. The catalytic optimal pH values for all the mutants remained unchanged with an identical value of 6.0.

DOI： 10.1271/bbb.63.1171

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROYAL SOC CHEMISTRY  

Site-directed mutagenesis experiments with W169F, W169H and W489F for the squalene-hopene cyclase, and the formation of 10 possessing the five-membered D-ring and a tetrahydrofuran moiety as the enzyme product of the analogue 8 with a hydroxy group, strongly suggest that a ring expansion reaction from the five- to the six-membered ring is responsible for the D-ring formation of hopene.

DOI： 10.1039/a806948d

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担当区分：筆頭著者   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  

An overexpression system for squalene-hopene cyclase (SHC) was constructed by using the pET3a vector, which is responsible for high expression with help from the strong T7 promoter when incorporated into E. coli BL21(DE3). Site-directed mutagenesis experiments prove that two amino acid residues of tryptophan and aspartic acid inside the QW-motif 5 resided as active sites.

DOI： 10.1271/bbb.62.407

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記述言語：日本語   出版者・発行元：芝浦工業大学連携推進部  

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記述言語：日本語   出版者・発行元：天然有機化合物討論会実行委員会  

&lt;p&gt;はじめに&lt;/p&gt;&lt;p&gt;我々は、天然物探索から新規C&lt;sub&gt;35&lt;/sub&gt;テルペンを発見して以来、C&lt;sub&gt;35&lt;/sub&gt;テルペンに着目して研究を行っている&lt;sup&gt;1-6)&lt;/sup&gt;。50,000種類を超えるテルペン類の中で、C&lt;sub&gt;35&lt;/sub&gt;テルペンには長らく特別な分類名がなかったが、直鎖状C&lt;sub&gt;35&lt;/sub&gt;イソプレノイドの環化を経る経路を酵素・遺伝子レベルで初めて証明し、「セスクアテルペン」と命名した&lt;sup&gt;2-5)&lt;/sup&gt;。本経路には、1990年代から数多く見出されてきたテルペン環化酵素の一次構造と類似性をもたない「新型」テルペン環化酵素（テトラプレニル-β-クルクメン合成酵素：TS）が関わることを明らかにした（Scheme 1）&lt;sup&gt;2-5)&lt;/sup&gt;。また、Bacillus megaterium由来のテトラプレニル-β-クルクメン環化酵素（TC）は、C&lt;sub&gt;35&lt;/sub&gt;とC&lt;sub&gt;30&lt;/sub&gt;の基質から各々４環と２環を形成する二機能性テルペン環化酵素であることを証明した&lt;sup&gt;2,3,6)&lt;/sup&gt;。異なる２種類のテルペンを生体内で生合成する初めてのテルペン環化酵素であった（Scheme 1）&lt;sup&gt;2,3,6)&lt;/sup&gt;。&lt;/p&gt;&lt;p&gt;今回、セスクアテルペン環化酵素であるTSおよびTCに関する研究をさらに展開し、テルペン創出経路を開拓したので報告する。&lt;/p&gt;&lt;p&gt; &lt;/p&gt;&lt;p&gt;１）TSホモログのゲノムマイニングによる新規セスタテルペン・新規トリテルペンの発見&lt;sup&gt;7)&lt;/sup&gt;&lt;/p&gt;&lt;p&gt;TSホモログは、様々なバクテリアにおいて機能未知遺伝子として存在している。今回、手始めに好アルカリ性のBacillus clausiiのもの（Bcl-TS）をターゲットにした。B. clausiiゲノムにおいてTSホモログが存在するにも関わらず、菌体から化合物1が検出されなかった。一方、テルペン類と考えられる未知脂質（5と6）がGC-MSによって検出されたため、それらを単離・構造決定した。その結果、新規非環状セスタテルペン（5）と新規非環状トリテルペン（6）であることが判明した。各々β-geranylfarneseneおよびβ-hexapreneと命名した。&lt;/p&gt;&lt;p&gt;B. clausiiゲノムにおいてBcl-TS以外にテルペン合成酵素ホモログが存在しないことから、Bcl-TSが化合物5と6の生合成に寄与していることが考えられた。Bcl-TSの大腸菌発現系を構築後、精製Bcl-TSによってGFPP（C&lt;sub&gt;25&lt;/sub&gt;）とHexPP（C&lt;sub&gt;30&lt;/sub&gt;）から各々5と6が生成されることをin vitroで証明した（Scheme 2）。&lt;/p&gt;&lt;p&gt;Table 1に示したように、化合物5と6は多くの好アルカリ性Bacillusに分布していた&lt;sup&gt;7)&lt;/sup&gt;。以前、B. alcalophilusのような好アルカリ性Bacillusはスクアレン3やデヒドロスクアレンを生産することが報告されていたが&lt;sup&gt;8)&lt;/sup&gt;、我々は化合物5と6が生産されていると訂正した。&lt;/p&gt;&lt;p&gt; 本研究によって、TSホモログがセスクアテルペン（C&lt;sub&gt;35&lt;/sub&gt;）だけでなくC&lt;sub&gt;25&lt;/sub&gt;やC&lt;sub&gt;30&lt;/sub&gt;のような様々なテルペン合成酵素のファミリーであることが判明した。今後も、TSホモログのゲノムマイニングによって多くの新規テルペンを発見できるのではないかと期待している。&lt;/p&gt;&lt;p&gt;２）TCによる&lt;/p&gt;&lt;p&gt;(View PDFfor the rest of the abstract.)&lt;/p&gt;

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記述言語：日本語   出版者・発行元：日本香料協会  

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記述言語：日本語   出版者・発行元：日本生物工学会  

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その他リンク： http://dl.ndl.go.jp/info:ndljp/pid/10536373

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記述言語：日本語   出版者・発行元：天然有機化合物討論会実行委員会  

<p>はじめに</p><p>我々は、天然物探索から新規C₃₅テルペンを発見して以来、C₃₅テルペンに着目して研究を行っている^1-6)。50,000種類を超えるテルペン類の中で、C₃₅テルペンには長らく特別な分類名がなかったが、直鎖状C₃₅イソプレノイドの環化を経る経路を酵素・遺伝子レベルで初めて証明し、「セスクアテルペン」と命名した^2-5)。本経路には、1990年代から数多く見出されてきたテルペン環化酵素の一次構造と類似性をもたない「新型」テルペン環化酵素（テトラプレニル-β-クルクメン合成酵素：TS）が関わることを明らかにした（Scheme 1）^2-5)。また、Bacillus megaterium由来のテトラプレニル-β-クルクメン環化酵素（TC）は、C₃₅とC₃₀の基質から各々４環と２環を形成する二機能性テルペン環化酵素であることを証明した^2,3,6)。異なる２種類のテルペンを生体内で生合成する初めてのテルペン環化酵素であった（Scheme 1）^2,3,6)。</p><p>今回、セスクアテルペン環化酵素であるTSおよびTCに関する研究をさらに展開し、テルペン創出経路を開拓したので報告する。</p><p> </p><p>１）TSホモログのゲノムマイニングによる新規セスタテルペン・新規トリテルペンの発見⁷⁾</p><p>TSホモログは、様々なバクテリアにおいて機能未知遺伝子として存在している。今回、手始めに好アルカリ性のBacillus clausiiのもの（Bcl-TS）をターゲットにした。B. clausiiゲノムにおいてTSホモログが存在するにも関わらず、菌体から化合物1が検出されなかった。一方、テルペン類と考えられる未知脂質（5と6）がGC-MSによって検出されたため、それらを単離・構造決定した。その結果、新規非環状セスタテルペン（5）と新規非環状トリテルペン（6）であることが判明した。各々β-geranylfarneseneおよびβ-hexapreneと命名した。</p><p>B. clausiiゲノムにおいてBcl-TS以外にテルペン合成酵素ホモログが存在しないことから、Bcl-TSが化合物5と6の生合成に寄与していることが考えられた。Bcl-TSの大腸菌発現系を構築後、精製Bcl-TSによってGFPP（C₂₅）とHexPP（C₃₀）から各々5と6が生成されることをin vitroで証明した（Scheme 2）。</p><p>Table 1に示したように、化合物5と6は多くの好アルカリ性Bacillusに分布していた⁷⁾。以前、B. alcalophilusのような好アルカリ性Bacillusはスクアレン3やデヒドロスクアレンを生産することが報告されていたが⁸⁾、我々は化合物5と6が生産されていると訂正した。</p><p> 本研究によって、TSホモログがセスクアテルペン（C₃₅）だけでなくC₂₅やC₃₀のような様々なテルペン合成酵素のファミリーであることが判明した。今後も、TSホモログのゲノムマイニングによって多くの新規テルペンを発見できるのではないかと期待している。</p><p>２）TCによる</p><p>(View PDFfor the rest of the abstract.)</p>

DOI： 10.24496/tennenyuki.56.0_Oral18

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記述言語：英語   掲載種別：書評論文，書評，文献紹介等  

To the best of my knowledge, only 19 cyclic and 8 linear C35 terpenes have been identified to date, and no family name was assigned to this terpene class until recently. In 2011, it was proposed that these C35 terpenes should be called sesquarterpenes. This review highlights the biosynthesis of two kinds of sesquarterpenes (C35 terpenes) that are produced via cyclization of a linear C35 isoprenoid in Bacillus and Mycobacterium species. In Bacillus species, a new type of terpene cyclase that has no sequence homology with any known terpene synthases, as well as a bifunctional terpene cyclase that biosynthesizes two classes of cyclic terpenes with different numbers of carbons as natural products, have been identified. On the other hand, in Mycobacterium species, the first bifunctional Z-prenyltransferase has been found, but a novel terpene cyclase and a unique polyprenyl reductase remain unidentified. The identification of novel enzyme types should lead to the discovery of many homologous enzymes and their products including novel natural compounds. On the other hand, many enzymes responsible for the biosynthesis of natural products have low substrate specificities in vitro. Therefore, to find novel natural products present in organisms, the multifunctionality of enzymes in the biosynthetic pathway of natural products should be analyzed.

DOI： 10.1271/bbb.130180

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記述言語：日本語   出版者・発行元：公益社団法人 日本農芸化学会  

DOI： 10.1271/kagakutoseibutsu.50.622

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記述言語：日本語   出版者・発行元：天然有機化合物討論会実行委員会  

In this study, mono- and pentacyclic C_<35> terpenes from Bacillus subtilis were biosynthesized via the cyclization of C_<35> isoprenoid by using purified enzymes, including the first identified new terpene cyclase, tetrapreny1-13-curcumene synthase, that shows no sequence homology to any of the known terpene cyclases. Based on these findings, we propose that these C_<35> terpenes are called the new family of "sesquarterpenes." This study demonstrated that a tetrapreny1-β-curcumene cyclase (TC) from B. subtilis, which was originally identified as a sesquarterpene cyclase converting a head-to-tail type of monocycle to a pentacycle, also cyclized a tail-to-tail type of linear squalene into a bicyclic triterpenol, 8α-Hydroxypolypoda-13,17,21-triene. The 8α-Hydroxypolypoda-13,17,21-triene was found to be a natural triterpene from B. megaterium, suggesting that the TC is bifunctional, cyclizing both tetrapreny1-α-curcumene and squalene in vivo. This is the first report describing the bifunctional terpene cyclase, which biosynthesizes 2 classes of cyclic terpenes with different numbers of carbons as natural products in the organism. Non-pathogenic Mycobacterium species also produce cyclic sesquarterpenes, which are biosynthesized via cyclization of Z-type C_<35> polyprenyl diphosphate. To provide deeper insight into the biosynthesis of sesquarterpenes, we carried out functional analyses of three Z-prenyltransferase homologues in M vanbaalenii identified by genomic analysis. Mvan_3822, a novel bi-functional Z-prenyltransferase, biosynthesizes C_<35>-heptaprenyl diphosphate as a main product from E,E-FPP and E,E,E-GGPP, but produces a C_<50>-decaprenyl diphosphate from GPP. Mvan_1705 is a novel Z,E,E-GGPP synthase. In addition, novel cyclic C_<35>-terpenes, 14E- and 14Z-dehydroheptaprenylcycline, were identified as minor metabolites in non-pathogenic Mycobacterium cells. Sesquarterpenes could be biosynthesized by two routes, in which E- and Z-geometric isomers of heptaprenyl diphosphate are produced from E,E-FPP and E,E,E-GGPP, and the prenylreductase responsible for the biosynthesis of sesquarterpenes may work to reduce both E- and Z-prenyl residues. The studies on sesquarterpenes promise to be an attractive field for expanding our understanding of the terpene world.

DOI： 10.24496/tennenyuki.53.0_97

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記述言語：日本語   出版者・発行元：天然有機化合物討論会実行委員会  

Lipid components from 12 nonpathogenic Mycobacterium species were analysed. A novel cyclic C_<35>-terpene, named heptaprenylcycline 1, was obtained from 3 species, while octahydroheptaprenol 2, which has 3 Z-double bonds, was obtained from 6 species. The amounts of 1 and 2 in the cultured cells increased after the 4- to 6-d stationary phase. The yield of 1 was considerably greater at a higher temperature of 37℃ than at an optimal growth temperature of 28℃, while that of 2 remained unchanged at all the temperatures (20-40℃). A feeding experiment with D-[1-^<13>C] glucose revealed that 1 was produced via isopentenyl diphosphate, which is a metabolite of glycolysis and the methylerythritol phosphate pathway. The conversion of octahydroheptaprenyl diphosphate 2-PP to 1 was successful by using the cell-free extracts of M. chlorophenolicum, demonstrating that 2-PP is the biosynthetic intermediate of 1. This is the first example of the biosynthesis of a natural terpene via the cyclisation of a linear C_<35>-isoprenoid. The substrate 2-PP has Z-type prenyl moieties; however, terpene cyclases usually employ E-type isoprenoids. The gene encoding the terpene cyclase that cyclises prenyl diphosphate containing Z-double bonds has not been reported hitherto. Despite a careful search using the FASTA3 program, we could not detect any gene homologous to the known diphosphate-triggered type of mono-, sesqui- and diterpene cyclases in the genome of M. vanbaalenii, the DNA sequence of which has recently been elucidated. This suggests that a novel type of terpene cyclase might exist in the nonpathogenic Mycobacterium species.

DOI： 10.24496/tennenyuki.50.0_517

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記述言語：日本語   出版者・発行元：天然有機化合物討論会実行委員会  

Based upon the genomic database of Mycobacterium tuberculosis H37Rv, the gene Rv3377c,which is a putative terpene cyclase, was amplified by PCR and ligated into BamH I/Hind III site of pET 22b(+), and expressed in E. coli. BL21(DE3). The expressed Rv3377c gene product is almost insoluble (inclusion body), but coexpression of Rv3377c and chaperon in E. coli afforded the gene product as an active form. The functionally expressed gene product had an enzyme activity only for geranylgeranyl diphosphate (GGPP), but inert to geranyl-pp (GPP), farnesyl-pp (FPP), squalene and oxidosqulaene. The structural analysis of the enzymatic product revealed that this enzyme encodes the diterpene cyclase for producing the halimane diterpene skeleton. We named tuberculosinol 1 for the enzyme product. The detailed investigation on the gene product indicated that the actual product by the Rv3377c enzyme was tuberculosinol diphosphate 2. Cyclic diterpene skeletons are usually constructed after removing the PP functional group. After the trials to find the dephosphorylating enzyme, it has turned out that the flanking gene Rv3378c is responsible for the dephosphorylation reaction of 2, yielding the hydroxylated product (isotuberculosinol 3) and 1 resulting from a water attack. Both of the Rv3377c and Rv3378c have been found only the pathogenic species, but not in the non-pathogenic one. Recently, Russell and coworkers reported that the survival of Mycobacterium tuberculosis in the macrophage was attenuated by the gene disruption Thus, the two genes may be closely related to the pathogenicity. We evaluated the effect of 1 on phagocytic activity against zymosan, a yeast cell wall component that produces inflammation, using human macrophage-like U937 cells. Fluorescence microscopic analysis revealed that the phagocytosis of FITC-labeled zymosan by U937 cells treated with 10μM of 1 was reduced by about 50% compared to the cells without treatment. We discuss the detailed enzyme characterization including the kinetic data and substrate specificities. The substrate specificity of the Rv3378c enzyme was remarkably broad as well as cyc2, which was found in Streptomyces griseolosporeus. The Rv3378c and cyc2 accepted copalyl-PP (CDP), ent-CDP and syn-CDP as substrate, leading to the successful construction of a variety of diterpene skeletons.

DOI： 10.24496/tennenyuki.48.0_19

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記述言語：日本語   出版者・発行元：新潟大学農学部  

我々はオキシドスクアレン-ラノステロール環化酵素(OLC)の触媒機構を解明するため、酵母の発現糸を構築することを計画した。しかし、野生型の酵母は自身のOLCを持っていることから、そのまま発現宿主として用いることはできない。したがって、 OLC欠損型の酵母変異株を取得することを目的に実験を行った。酵母OLC欠損型二倍体から胞子形成を経て一倍体を分離した。得られた株(3-5-1)について、抗生物質に対する抵抗性の調査や脂質成分の分析そしてOLCの酵素活性の検定を行った。全ての結果は、 3-5-1株がOLC欠損型一倍体であることを示唆していた。今後、 3-5-1株は外来OLCの発現宿主として利用することができると考えられる。In order to investigate the catalytic mechanism of oxidosqualene-lanosterol cyclase (OLC), we have planed to construct the expression systenl of OLC in yeast Saccharomyces cerevtsiae. However, wild-type yeast can not be used as the expression host of OLC from o山er organism, because yeasts naturally have its own OLC. Therefore, we made experiments to obtain the yeast OLC deficient mutant. The OLC deficient haploid was separated from the OLC deficient diploid after the sporulation. The obtained microorganism, named 3-5-1, was confirmed to be OLC deficient haploid by checking the resistance for antibiotics, analyzing the components of lipid and assaying the OLC activity. The strain, named 3-5-1, may be able to be used as the expression host of OLC from other organism in future.

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その他リンク： http://hdl.handle.net/10191/25024

記述言語：日本語   出版者・発行元：新潟大学農学部  

古くから、オキシドスクアレン-ラノステロール環化酵素(OLC)の研究が成されてきているが、酵素アミノ酸残基レベルでの触媒機構こついては不明な点が数多く残されたままである。その解明には、異種細胞での高発現系の構築が必須であり、当面の課題となってきている。我々は、大腸菌におけるCandida albicans由来OLCの高発現系の構築を目的として実験を行った。まず、天然型のOLCを大腸菌で発現させた。しかし、不溶性であり、酵素活性も検出できなかった。次に、他の酵素の研究における可溶化の成功例やスクアレンーホペン環化酵素の研究を通して得た知見を参考にして、 TrxまたはDsb融合型、 N末端削除型、 QWモチーフ改変型の合計9種頬のOLCを大腸菌で発現させてみた。しかし、不溶性のままであり、酵素活性も検出できなかった。様々な方法によってOLCを大腸菌で発現させたにも関わらず、不溶性の問題を解決できなかったことから、 OLCの発現宿主には酵母や昆虫細胞などの真核生物を利用する必要があることが示唆された。

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その他リンク： http://hdl.handle.net/10191/25023

記述言語：日本語   出版者・発行元：公益社団法人 日本農芸化学会  

DOI： 10.1271/nogeikagaku1924.77.430

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記述言語：英語   出版者・発行元：ROYAL SOC CHEMISTRY  

Rapid progress on the catalytic mechanism and substrate recognition by squalene-hopene cyclase, which has occurred only in the last several years, is reported. A series of site-directed mutation experiments and some squalene analogues have provided deep insight into the polycyclization mechanism and catalytic sites in conjunction with the information from X-ray crystal data.

DOI： 10.1039/b108995c

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記述言語：日本語   出版者・発行元：天然有機化合物討論会実行委員会  

Squalene cyclization mechanism and the active sites of the cyclase are discussed on the basis of the site-directed mutagenesis experiments. 1. DXDDTA motif: the initiation of the polycyclization and stabilization of carobocation intermediate of the initialy formed A-ring through the carboxylate anion of D377. 2. Phe365 stabilizes the C-8 carbocation intermediate of A/B-fused ring system through cation/π interaction. This interaction was confirmed by constructing the mutants F365Y and F365W; these mutants accelerated the reaction velocity and the significant lowering of activation energy for the polycyclization reaction. Tyr420 is also responsible for the formation of B-ring. 3. Phe 601is crucial for the construction of the 6-membered C/D-ring system. The mutant F601A produced the partially cyclized tricyclic 6/6/5-fused13,14 and tetracyclic 6/6/6/5-fused 18. These products could be formed by a Markovnikov closure and indicated the involvements of ring-expansion processes from the 5-membered into the 6-membered ring system for the construction of the C/D-ring system. Compound 18 was also isolated from the mutant W169F and W169H. The presumed carbocationic intermediates 12 and 17 was trapped by using the substrate analogues 27 and 32(34), respectively, with a high nucleophilic hydroxyl group, resulting in the formation of 31 and 35. Thus, we propose that the polycyclization mechanism proceeds via two ring-expansion steps for the formation of C- and D-rings, leading to the formation of anti-Markovnikov adduct 2. 4. The mutant I261A produced a series of carbocationic intermeidates 6/6/5-, 6/6/6/5-, and 6/6/6/6-fused ring systems, giving further insight into the polycycliation mechansim. In the enzymatic products, unnatural natural products are included. 5. Phe 605 would stabilize the cations of 17, 19 and 20 through cation-π interaction, as verified by the isolations of 6/6/6/5- and 6/6/6/6/5-ring skeletons. 6. The methyl at C10-posisiton of 1 is crucial to the correct folding in the active center, this being demonstrated by the substrate analouge 40. The proposed cyclization mechanism has been verified both by the kinetic measurments and by the isolation of the differently cyclized products resulting from each stage of the carbocationic intermediates. Alteration of the cyclase active sites afforded multiple triterpenes in addtion to the understanding of the fundamental issues of squalene cyclization mechanism, suggesting the possibility that we will be able to generate novel triterpenes (unnatural natural products) by rational genetic engineering of squalene cyclase.

DOI： 10.24496/tennenyuki.41.0_199

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記述言語：日本語   出版者・発行元：天然有機化合物討論会実行委員会  

There has been remarkable advances in the studies of oxidosqualene and squalene cyclases in the past ten years. First, complete purifications of the enzymes have been attained from several species such as vertebrates, plant, yeast and bacterium, irrespective of unstable and membrane-bound nature. Second, application of cDNA cloning technique to the cyclases have succeeded in determining the alignments of amino acids from various biological sources including human. Third, the substrate analogues including the suicide inhibitors have made a great contribution to the polycyclization mechanism. In this symposium, we report a definitive evidence that the polycyclization proceeds via the discrete carbocationic intermediate formed during the cyclization, but not via a concerted manner as proposed before; substitution of methyls with ethyl groups at 10- and 15-positions 6 halted the enzymic reaction at the monocyclic stage 7 and 8. Substrate analogue 9 with ethyl group at 15-position afforded 10 (normal cyclization) and 11 (tricyclic 6/6/5), the latter being formed under the control of Markovnikov rule. This is in contrast to the protosterol cation, which is formed under anti-Markovnikov control. Thus, oxidosqualene would be converted via the 5-membered intermediate 12 under the control of Markovnikov rule, which then undergoes a ring expansion to form 6-membered C-ring. Mechanisms of the formation of tetrahymanol and hopene from squalene itself are discussed, especially on the terminal cyclization (E-ring formation). Substrate analogs 14, 15, 18, 22 have demonstrated the cyclization mechanisms as follows: for tetrahymanol cyclase, the terminal cyclization proceeds by the process of stereoelectronic control, suggesting little participation of the enzyme, while hopene cyclase strongly binds to the terminal methyl groups to form 5-membered E-ring. From our studies, methyl groups of the substrate seem to bind the cyclase enzymes and the substrates were then subjected to the folding of chair-boat or chair-chair inside the enzymes leading to production of the desired triterpene skeletons. Over-expression of hopene cyclase was achieved successfully and the point mutations of amino acids in QW motif proved that the functions of D and W are crucial for the enzyme activity.

DOI： 10.24496/tennenyuki.39.0_145

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記述言語：日本語   出版者・発行元：社団法人日本農芸化学会  

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

<p>はじめに</p><p>我々は、天然物探索から新規C₃₅テルペンを発見して以来、C₃₅テルペンに着目して研究を行っている^1-6)。50,000種類を超えるテルペン類の中で、C₃₅テルペンには長らく特別な分類名がなかったが、直鎖状C₃₅イソプレノイドの環化を経る経路を酵素・遺伝子レベルで初めて証明し、「セスクアテルペン」と命名した^2-5)。本経路には、1990年代から数多く見出されてきたテルペン環化酵素の一次構造と類似性をもたない「新型」テルペン環化酵素（テトラプレニル-β-クルクメン合成酵素：TS）が関わることを明らかにした（Scheme 1）^2-5)。また、Bacillus megaterium由来のテトラプレニル-β-クルクメン環化酵素（TC）は、C₃₅とC₃₀の基質から各々４環と２環を形成する二機能性テルペン環化酵素であることを証明した^2,3,6)。異なる２種類のテルペンを生体内で生合成する初めてのテルペン環化酵素であった（Scheme 1）^2,3,6)。</p><p>今回、セスクアテルペン環化酵素であるTSおよびTCに関する研究をさらに展開し、テルペン創出経路を開拓したので報告する。</p><p> </p><p>１）TSホモログのゲノムマイニングによる新規セスタテルペン・新規トリテルペンの発見⁷⁾</p><p>TSホモログは、様々なバクテリアにおいて機能未知遺伝子として存在している。今回、手始めに好アルカリ性のBacillus clausiiのもの（Bcl-TS）をターゲットにした。B. clausiiゲノムにおいてTSホモログが存在するにも関わらず、菌体から化合物1が検出されなかった。一方、テルペン類と考えられる未知脂質（5と6）がGC-MSによって検出されたため、それらを単離・構造決定した。その結果、新規非環状セスタテルペン（5）と新規非環状トリテルペン（6）であることが判明した。各々β-geranylfarneseneおよびβ-hexapreneと命名した。</p><p>B. clausiiゲノムにおいてBcl-TS以外にテルペン合成酵素ホモログが存在しないことから、Bcl-TSが化合物5と6の生合成に寄与していることが考えられた。Bcl-TSの大腸菌発現系を構築後、精製Bcl-TSによってGFPP（C₂₅）とHexPP（C₃₀）から各々5と6が生成されることをin vitroで証明した（Scheme 2）。</p><p>Table 1に示したように、化合物5と6は多くの好アルカリ性Bacillusに分布していた⁷⁾。以前、B. alcalophilusのような好アルカリ性Bacillusはスクアレン3やデヒドロスクアレンを生産することが報告されていたが⁸⁾、我々は化合物5と6が生産されていると訂正した。</p><p> 本研究によって、TSホモログがセスクアテルペン（C₃₅）だけでなくC₂₅やC₃₀のような様々なテルペン合成酵素のファミリーであることが判明した。今後も、TSホモログのゲノムマイニングによって多くの新規テルペンを発見できるのではないかと期待している。</p><p>２）TCによる</p><p>(View PDFfor the rest of the abstract.)</p>

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記述言語：日本語  

In this study, mono- and pentacyclic C_<35> terpenes from Bacillus subtilis were biosynthesized via the cyclization of C_<35> isoprenoid by using purified enzymes, including the first identified new terpene cyclase, tetrapreny1-13-curcumene synthase, that shows no sequence homology to any of the known terpene cyclases. Based on these findings, we propose that these C_<35> terpenes are called the new family of "sesquarterpenes." This study demonstrated that a tetrapreny1-β-curcumene cyclase (TC) from B. subtilis, which was originally identified as a sesquarterpene cyclase converting a head-to-tail type of monocycle to a pentacycle, also cyclized a tail-to-tail type of linear squalene into a bicyclic triterpenol, 8α-Hydroxypolypoda-13,17,21-triene. The 8α-Hydroxypolypoda-13,17,21-triene was found to be a natural triterpene from B. megaterium, suggesting that the TC is bifunctional, cyclizing both tetrapreny1-α-curcumene and squalene in vivo. This is the first report describing the bifunctional terpene cyclase, which biosynthesizes 2 classes of cyclic terpenes with different numbers of carbons as natural products in the organism. Non-pathogenic Mycobacterium species also produce cyclic sesquarterpenes, which are biosynthesized via cyclization of Z-type C_<35> polyprenyl diphosphate. To provide deeper insight into the biosynthesis of sesquarterpenes, we carried out functional analyses of three Z-prenyltransferase homologues in M vanbaalenii identified by genomic analysis. Mvan_3822, a novel bi-functional Z-prenyltransferase, biosynthesizes C_<35>-heptaprenyl diphosphate as a main product from E,E-FPP and E,E,E-GGPP, but produces a C_<50>-decaprenyl diphosphate from GPP. Mvan_1705 is a novel Z,E,E-GGPP synthase. In addition, novel cyclic C_<35>-terpenes, 14E- and 14Z-dehydroheptaprenylcycline, were identified as minor metabolites in non-pathogenic Mycobacterium cells. Sesquarterpenes could be biosynthesized by two routes, in which E- and Z-geometric isomers of heptaprenyl diphosphate are produced from E,E-FPP and E,E,E-GGPP, and the prenylreductase responsible for the biosynthesis of sesquarterpenes may work to reduce both E- and Z-prenyl residues. The studies on sesquarterpenes promise to be an attractive field for expanding our understanding of the terpene world.

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記述言語：日本語  

Lipid components from 12 nonpathogenic Mycobacterium species were analysed. A novel cyclic C_<35>-terpene, named heptaprenylcycline 1, was obtained from 3 species, while octahydroheptaprenol 2, which has 3 Z-double bonds, was obtained from 6 species. The amounts of 1 and 2 in the cultured cells increased after the 4- to 6-d stationary phase. The yield of 1 was considerably greater at a higher temperature of 37℃ than at an optimal growth temperature of 28℃, while that of 2 remained unchanged at all the temperatures (20-40℃). A feeding experiment with D-[1-^<13>C] glucose revealed that 1 was produced via isopentenyl diphosphate, which is a metabolite of glycolysis and the methylerythritol phosphate pathway. The conversion of octahydroheptaprenyl diphosphate 2-PP to 1 was successful by using the cell-free extracts of M. chlorophenolicum, demonstrating that 2-PP is the biosynthetic intermediate of 1. This is the first example of the biosynthesis of a natural terpene via the cyclisation of a linear C_<35>-isoprenoid. The substrate 2-PP has Z-type prenyl moieties; however, terpene cyclases usually employ E-type isoprenoids. The gene encoding the terpene cyclase that cyclises prenyl diphosphate containing Z-double bonds has not been reported hitherto. Despite a careful search using the FASTA3 program, we could not detect any gene homologous to the known diphosphate-triggered type of mono-, sesqui- and diterpene cyclases in the genome of M. vanbaalenii, the DNA sequence of which has recently been elucidated. This suggests that a novel type of terpene cyclase might exist in the nonpathogenic Mycobacterium species.

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