Koji Ichinose

A mutation in actVI-ORF1, which controls C-3 reduction in actinorhodin biosynthesis by Streptomyces coelicolor, was complemented by gra-ORF5 and -ORF6 from the granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tu22. It is hypothesized that, while gra-ORF5 alone is a ketoreductase for C-9, gra-ORF6 gives the enzyme regiospecificity also for C-3.

1,3,6,8-Tetrahydroxynaphthalene (T4HN) is an aromatic polyketide, serving as a general precursor of fungal melanin. Melanin biosynthesis involves two consecutive deoxygenations of T4HN, consisting of the reduction of a phenolic carbon followed by dehydration. The first reduction to produce scytalone was studied in a biomimetic reduction with sodium borohydride. The reduction required a strong alkaline condition, leading to the tautomerization of T4HN to a reactive species whose structure was clarified bit NMR spectroscopy.

Background: Two deoxysugar glycosyltransferases (GTs), UrdGT1b and UrdGT1c, involved in urdamycin biosynthesis share 91% identical amino acids. However, the two GTs show different specificities for both nucleotide sugar and acceptor substrate. Generally, it is proposed that GTs are two-domain proteins with a nucleotide binding domain and an acceptor substrate site with the catalytic center in an interface cleft between these domains. Our work aimed at finding out the region responsible for determination of substrate specificities of these two urdamycin GTs. Results: A series of 10 chimeric GT genes were constructed consisting of differently sized and positioned portions of urdGT1b and urdGT1c. Gene expression experiments in host strains Streptomyces fradiae Ax and XTC show that nine of 10 chimeric GTs are still functional, with either UrdGT1b- or UrdGT1c-like activity. A 31 amino acid region (aa 52-82) located close to the N-terminus of these enzymes, which differs in 18 residues, was identified to control both sugar donor and acceptor substrate specificity. Only one chimeric gene product of the 10 was not functional. Targeted stepwise alterations of glycine 226 (G226R, G226S, G226SR) were made to reintroduce residues conserved among streptomycete GTs. Alterations G226S and G226R restored a weak activity, whereas G226SR showed an activity comparable with other functional chimeras. Conclusions: A nucleotide sugar binding motif is present in the C-terminal moiety of UrdGT1b and UrdGT1c from S. fradiae. We could demonstrate that it is an N-terminal section that determines specificity for the nucleotide sugar and also the acceptor substrate. This finding directs the way towards engineering this class of streptomycete enzymes for antibiotic derivatization applications. Amino acids 226 and 227, located outside the putative substrate binding site, might be part of a larger protein structure, perhaps a solvent channel to the catalytic center. Therefore, they could play a role in substrate accessibility to it. © 2001 Elsevier Science Ltd.

バイオミデフィア2001連載の一環として、放線菌として初めて全ゲノム配列の解読が終了した<I>Streptomyces coeliclor</I>A3(2)と、本菌株が生産するpH感受性色素性抗生物質アクチノロジンについて紹介した記事

Background: Streptomyces fradiae is the principal producer of urdamycin A. The antibiotic consists of a polyketide-derived aglycone, which is glycosylated with four sugar components, 2X D-olivose (first and last sugar of a C-glycosidically bound trisaccharide chain at the g-position), and 2 x L-rhodinose (in the middle of the trisaccharide chain and at the 12b-position). Limited information is available about both the biosynthesis of D-olivose and L-rhodinose and the influence of the concentration of both sugars on urdamycin biosynthesis. R Results: To further investigate urdamycin biosynthesis, a 5.4 kb section of the urdamycin biosynthetic gene cluster was sequenced. Five new open reading frames (ORFs) (urdZ3, urdQ, urdR, urdS, urdT) could be identified each one showing significant homology to deoxysugar biosynthetic genes. We inactivated four of these newly allocated ORFs (urdZ3, urdQ, urdR, urdS) as well as urdZ1, a previously found putative deoxysugar biosynthetic gene. Inactivation of urdZ3, urdQ and urdZ1 prevented the mutant strains from producing L-rhodinose resulting in the accumulation of mainly urdamycinone B. Inactivation of urdR led to the formation of the novel urdamycin M, which carries a C-glycosidically attached D-rhodinose at the 9-position. The novel urdamycins N and O were detected after overexpression of urdGT1c in two different chromosomal urdGT1c deletion mutants. The mutants lacking urdS and urdQ accumulated Various known diketopiperazines. Conclusions: Analysis of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster revealed a widely common biosynthetic pathway leading to D-olivose and L-rhodinose. Several enzymes responsible for specific steps of this pathway could be assigned. The pathway had to be modified compared to earlier suggestions. Two glycosyltransferases normally involved in the C-glycosyltransfer of D-olivose at the 9-position (UrdGT2) and in conversion of 100-2 to urdamycin G (UrdGT1c) show relaxed substrate specificity for their activated deoxysugar co-substrate and their alcohol substrate, respectively. They can transfer activated D-rhodinose (instead of D-olivose) to the B-position, and attach L-rhodinose to the 4A-position normally occupied by a D-olivose unit, respectively.

NMR and MS spectroscopy elucidated the structure of a novel shunt product isolated from a disruptant of the actVI-ORFA gene involved in the biosynthesis of actinorhodin (ACT) in Streptomyces coelicolor A3(2). The product, 1,4-naphthoquinone-8-hydroxy-3-[(3S)-acetoxybutyric acid], was found without complete abolition of ACT production, implying that the ORFA product might stabilise the multicomponent, type II PKS complex and perhaps assist the chemically spontaneous dehydration of a hemiketal intermediate for efficient pyran ring formation. (C) 2000 Elsevier Science Ltd. All rights reserved.

The actVI genetic region of Streptomyces coelicolor A3(2) is part of the biosynthetic gene cluster of actinorhodin (ACT), the act cluster, consisting of six ORFs: ORFB, ORFA, ORF1, ORF2, ORF3, ORF4. A newly devised method of ACT detection with a combination of HPLC and LC/MS was applied to the analysis of the disruptants of each ORE ACT was produced by those of ORFB, ORFA, ORF3, and ORF4. Instead of ACT, the ORF1 disruptant produced 3,8dihydroxy-1-methylanthraquinone-2-carboxylic acid (DMAC) and aloesaponarin II as shunt products. The ORF2 disruptant gave 4-dihydro-9-hydrsxy-1-methyl-10-oxo-3-H-naphtho-[2,3-c]-pyran-3-(S)-acetic acid, (S)-DNPA. These results support our previous proposal for stereospecific pyran ring formation in the biosynthesis of ACT, most importantly suggesting that the actVI-ORF2 product would recognize (S)-DNPA as a substrate for stereospecific reduction at C-15. The disruptant of ORFA produced (S)-DNPA together with ACT, suggesting that actVI-ORFA might play a role such as stabilising the multicomponent, type II PKS complex.

Potential "reagents" for the enantioselective reduction, and other biotransformations, of beta-keto-esters result from the genetic engineering of <I>Streptomyces coelicolor </I>A3(2). For example, incubation of the N-acetylcysteamine thioester 1 with the recombinant strain CH999/pIJ5675 followed by treatment with MeOH/HCl gave the lactone 2 as essentially a single enantiomer.

多段階酵素反応を必要とする天然有機化合物の生合成では，生合成酵素遺伝子群の協調的な発現制御が必要と考えられるが，近年これに関与する制御遺伝子が生合成遺伝子クラスター中に続々と発見され，生合成遺伝子の特異的な転写活性化を説明するモデルが提出された。このモデルの特徴は大腸菌OmpRに代表される細菌の二成分制御系の転写因子に存在するDNA認識機構と類似したものであることで、本稿はこの研究発展の端緒になったゲノム研究基軸放線菌 <I>Streptomyces coelicolor</I> の最近の話題を導入部に，筆者らが研究しているポリケタイド化合物の生合成を例に経路特異的転写活性化機構を解説し，その物質生産系への応用例にまで言及した総説である。（和文）

ビタミンB<SUB>12</SUB>は１９員環コリン骨格にヌクレオチドループが結合した最も複雑な天然大環状色素である。コリン骨格の形成にはポリフィリン類との共通の生合成中間体であるウロポルフィリンIIIからの特異的経路が関与する。ここで２０員環からの環縮合が生じるが、ここに還元過程が含まれるか否かを、立体特異的同位体標識したNAD(P)H（論文3.参照）を用いたトレーサー実験により検討した。従来は還元過程は含まないとされていた嫌気性B<SUB>12</SUB>生産細菌においても還元過程が含まれることを証明することができ、コリン環生成に関する普遍的生合成経路の存在を示唆する結果となった。市瀬は第一著者として本論文の結論に関わるの最重要実験に関与した。

ステロイドサポニン生合成の最終段階で機能する特異的な・-グルコシダーゼとして <I>Costus speciosus </I>（オオホザキアヤメ: Costaceae）由来の酵素(CSF26G)を取りあげ、植物での代謝工学的応用の試みとして、異種植物に導入し発現を試みた。遺伝子導入にはバイナリーベクターpBI121を利用してCauliflower Mosaic Virus 35S Promoter下流に目的cDNAを接続して発現ベクターを構築した。これを <I>Agrobacterium tumefaciens </I>に導入し、得られた形質転換体をタバコ に感染させた。カルスから再生したカナマイシン耐性のクローン幼植物体８株について、ゲノムDNAを抽出し、PCRにて全長遺伝子の組み込みが確認できたクローンを用いて無細胞粗酵素液を調製した。本酵素液について、原植物から単離報告のあるフロスタン型配糖体プロトディオスシンを基質として供したところ、対応するスピロスタン型配糖体ディオスシンを与える酵素活性を形質転換植物由来の粗酵素液にも確認することができた。市瀬は第一著

Pyran ring formation in the biosynthesis of actinorhodin in Streptomyces coelicolor A3(2) was studied using the act cluster deficient strain, CH999, carrying pRM5-based plasmids harbouring combinations of the actVI genes. The strain, CH999/pIJ5660 (pRM5 + actVI-ORF1), produced a chiral intermediate, (S)-DNPA, suggesting that the actVI-ORF1 product is a reductase determining the C-3 stereochemical centre. (C) 1999 Elsevier Science Ltd. All rights reserved.

Plant oxidosqualene cyclase (OSC) is classified into two types depeneding on its catalytic mechanisms and products: cycloartenol synthase and triterpene synthase. A cDNA probably encoding OSC was cloned from bulbs of Allium macrostemon by RT-PCR. The cDNA contains a 2289-bp open reading frame, encoding 762 amino acids. A higher degree of homology (73%) was found for the gene product with the known cycloartenol synthases than with the triterpene synthases (51-55%), indicating its most likely function as cycloartenol synthase. This is the first cloning example of a putative cDNA encoding OSC from a monocotyledonous plant.

Benzoisochromanequinone (BIQ) antibiotics are a class of arom. polyketides produced by <I>Streptomyces</I> spp. A polyketide synthase (PKS, Type II) is involved in the formation of each BIQ chromophore. The first PKS genes were cloned in <I>S. coelicolor </I>A3(2)?, producer of the typical BIQ antibiotic, actinorhodin; sequence anal. of the PKS genes revealed their clustering with all the other relevant biosynthetic genes (the act cluster). This review describes the genes from the act and gra (granaticin biosynthetic gene cluster in<I> S. violaceoruber </I>Tu22) clusters involved in post-?PKS modifying (tailoring) steps to complete BIQ biosynthesis.

Introduction: The granaticins are members of the benzoisochromanequinone class of aromatic polyketides, the best known member of which is actinorhodin made by Streptomyces coelicolor A3(2). Genetic analysis of this class of compounds has played a major role in the development of hypotheses about the way in which aromatic polyketide synthases (PKSs) control product structure. Although the granaticin nascent polyketide is identical to that of actinorhodin, post-PKS steps involve different pyran-ring stereochemistry and glycosylation. Comparison of the complete gene clusters for the two metabolites is therefore of great interest. Results: The entire granaticin gene cluster (the gra cluster) from Streptomyces violaceoruber Tu22 was cloned on either of two overlapping cosmids and expressed in the heterologous host, Streptomyces coelicolor A3(2), strain CH999. Chemical analysis of the recombinant strains demonstrated production of granaticin, granaticin B, dihydrogranaticin and dihydrogranaticin B, which are the four known metabolites of S. violaceoruber. Analysis of the complete 39,250 base pair sequence of the insert of one of the cosmids, pOJ466-22-24, revealed 37 complete open reading frames (ORFs), 15 of which resemble ORFs from the act (actinorhodin) gene cluster of S. coelicolor A3(2). Among the rest, nine resemble ORFs potentially involved in deoxysugar metabolism from Streptomyces spp. and other bacteria, and six resemble regulatory ORFs. Conclusions: On the basis of these resemblances, putative functional assignments of the products of most of the newly discovered ORFs were made, including those of genes involved in the PKS and tailoring steps in the biosynthesis of the granaticin aglycone, steps in the deoxy sugar pathway, and putative regulatory and export functions.

Biosyntheti studies on steroido saponins using a monocot plant, <I>Allium macrostemon</I>, and its related studies are summarized in this proceeding.

Six open reading frames (ORFs) were identified by DNA sequencing of 5.7 kilobase pairs at the left end of the act cluster (the so-called ''actVI region''), in the order: ORFB, ORFA, ORF1, ORF2, ORF3, ORF4, ORF1-4 are transcribed rightward and in the same direction as the ORFs of the actVA region which lies to the right of the actVI region, whereas ORFA and ORFB run in the opposite direction. By complementation of mutants and gene disruption of the wild type strain, the two previously genetically characterized actVI mutations were assigned to ORF1. Although disruption of ORFB and ORF4, using phi C31 derivatives, did not cause any obvious change in actinorhodin production, defects in actinorhodin synthesis were obtained by insertional inactivation of ORFA, ORF1, ORF2, or ORF3. RNA analysis within the ORF1/ORFA intergenic region showed overlapping divergent promoters, at least one of which is under the control of the actII-ORF4 gene product, the transcriptional activator of the act cluster. Data base searches with the deduced products of ORFB and ORF3 failed to show any significant similarities with other known proteins. The deduced product of ORFA strongly resembles those of genes of unknown function from Saccharopolyspora hirsuta and Streptomyces roseofulvus, located within polyketide synthase clusters. The ORF1 product strongly resembles beta-hydroxyacyl-CoA dehydrogenases of bacteria and mammals and the ORF2 and ORF4 products resemble each other and enoyl reductases from bacteria, animals, and plants, with a highly conserved cofactor-binding domain. These findings strongly suggest that the actVI region is involved in catalyzing reduction processes that determine the two stereochemical configurations at C-3/C-15 during actinorhodin biosynthesis. A scheme is proposed for the middle steps of the biosynthesis, that is formation of the pyran ring, leading to the benzoisochromanequinone structure.

[1,10,20-13C3]Uro'gen III is unambiguously synthesised for enzymic conversion into precorrin-4 isolated after aerial oxidation as two epimers of Factor IV, the 13C NMR spectra of which rigorously confirm the presence of a C-1 acetyl group in precorrin-4; attachment of the fourth methyl group at C-17 of precorrin-4 is also confirmed by related 13C-labelling experiments.

Two enzyme reactions involved in the post-aromatic deoxygenation process of fungal melanin and pigments were studied from the viewpoint of enzymic reduction of aromatic rings. Hydroxynaphthalene reductase that catalyzes reduction of the aromatic rings of 1,3,6,8-tetrahydroxynaphthalene and 1,3,8-trihydroxynapthalene was partially purified from Phialophora lagerbergii and characterized. Emodin deoxygenase of Pyrenochaeta terrestris that catalyzes deoxygenation of emodin to afford chrysophanol was found to be resolved into two protein fractions with DEAE-cellulose column. The two protein fractions acted synergistically in regard to emodin deoxygenase activity.

ニコチンアミドアデニンジヌクレオチド(NAD)またはそのリン酸化体(NADP)は生体内の酸化還元反応に関わる補酵素として普遍的に利用されている。これらの還元型であるNAD(P)Hはニコチンアミドの４位の水素をハイドライドとして供与することにより還元反応が進行する。供与可能な２つのハイドライドはプロキラル水素として酵素により厳密に区別されるが、その特異性を解析するためには一方のハイドライドを特異的に同位体標識したNAP(P)Hの調製が不可欠である。立体特異性が既知のアルコールデヒドロゲナーゼと酸化剤ピロロキノリンキノンを利用し、[4 <I>R</I>-<SUP>3</SUP>H]NAD(P)Hと対応する4<I>S</I>-異性体の４種類全てを効率的に調製する方法を開発した。調製した補酵素はビタミンB<SUB>12</SUB>生合成に含まれる還元反応の一つを解明することに利用した（論文６．参照）。市瀬は第一著者として本論文に関わる全ての実験を担当し、論文作成全般にも関わった。

Labelling experiments with 2H and 3H prove that as cobyrinic acid is biosynthesised by the anaerobic bacterium <I>Propionibacterium shermanii</I>, its H-19 is derived from HR(and not HS) at C-4 of a reduced nicotinamide cofactor, so demonstrating that the biosynthetic pathway involves a reduction step.

Summary of the mechanistic studies on the biomimetic reduction of 1,3,6,8-tetrahydroxynaphthalene in related to the melanin biosynthesis in phytopathogenic fungi, using NMR spectroscopy.

One-step chemical conversion of flavanones into isoflavones by use of thallium trinitrate (TIN) is reported, and the mechanism of a 2,3-aryl migration in this reaction is discussed in relation to <I>in vivo </I>rearrangement process of flavanone precursors in the isoflavone biosynthesis.

Two new pterocarpans named sophoracarpans A and B were isolated from <I>Sophora tomentosa</I> L. (Leguminosae), and their respective structures have been elucidated.