Co-reporter:Koji Narita, Hajime Sato, Atsushi Minami, Kosei Kudo, Lei Gao, Chengwei Liu, Taro Ozaki, Motoichiro Kodama, Xiaoguang Lei, Tohru Taniguchi, Kenji Monde, Mami Yamazaki, Masanobu Uchiyama, and Hideaki Oikawa
Organic Letters December 15, 2017 Volume 19(Issue 24) pp:6696-6696
Publication Date(Web):November 29, 2017
DOI:10.1021/acs.orglett.7b03418
Heterologous expression of four clade-A bifunctional terpene synthases (BFTSs), giving di/sesterterpenes with unique polycyclic carbon skeletons such as sesterfisherol, enabled the isolation of the sesterterpenes Bm1, Bm2, Bm3, and Pb1. Their structures suggested that formation of the products occurs via various diastereomeric carbocation intermediates, allowing the proposal that clade-A BFTSs catalyze three-step cyclizations using several stereofacial combinations of allylic cation–olefin pairs in the corresponding intermediates to generate various stereoisomers.
Co-reporter:Tomoshige Hiratsuka;Hideaki Suzuki;Atsushi Minami
Organic & Biomolecular Chemistry 2017 vol. 15(Issue 5) pp:1076-1079
Publication Date(Web):2017/02/01
DOI:10.1039/C6OB02675C
Jawsamycin is a polyketide–nucleoside hybrid with a unique polycyclopropane moiety on a single polyketide chain. The unexpected isolation of cyclopropane deficient jawsamycin analogs allowed us to propose a stepwise cyclopropanation mechanism for the enzymatic synthesis of this polyketide. The concise timing of the cyclopropanation could be regulated by a delicate balance between reaction rates of the condensation and cyclopropanation reactions.
Co-reporter:Chengwei Liu, Atsushi Minami, Tohru Dairi, Katsuya Gomi, Barry Scott, and Hideaki Oikawa
Organic Letters 2016 Volume 18(Issue 19) pp:5026-5029
Publication Date(Web):September 15, 2016
DOI:10.1021/acs.orglett.6b02482
The late-stage biosynthetic pathway of the indole diterpene shearinine involving four enzymatic reactions (JanQDOJ) was elucidated by an efficient heterologous expression system using Aspergillus oryzae. Key oxidative cyclization, forming a characteristic A/B bicyclic shearinine core by flavoprotein oxidase, was studied using a substrate analogue and a buffer containing H218O. These experimental data provided evidence that JanO catalyzes two-step oxidation via a hydroxylated product and that the JanO reaction involves the hydride-transfer mechanism.
Co-reporter:Koji Narita, Ryota Chiba, Atsushi Minami, Motoichiro Kodama, Isao Fujii, Katsuya Gomi, and Hideaki Oikawa
Organic Letters 2016 Volume 18(Issue 9) pp:1980-1983
Publication Date(Web):April 26, 2016
DOI:10.1021/acs.orglett.6b00552
Heterologous expression of four candidate genes found in ophiobolin gene clusters from three fungal strains was employed to elucidate the late-stage biosynthetic pathway of phytotoxin ophiobolin. Expression of oblBAc (cytochrome P450) from the cryptic gene cluster gave unexpected products, and that of oblBBm/oblBEv from the gene cluster of ophiobolin producers, with oblDBm as the transporter, yielded intermediate ophiobolin C through an unusual four-step oxidation process. The observation made in this study may provide a useful guideline for the elucidation of genuine biosynthetic pathways of natural products.
Co-reporter:Atsushi Minami and Hideaki Oikawa
The Journal of Antibiotics 2016 69(7) pp:500-506
Publication Date(Web):June 15, 2016
DOI:10.1038/ja.2016.67
Frequent occurrence of [4+2] adducts in the secondary metabolites suggested involvement of Diels–Alderases (DAases) in their biosynthesis. However, a limited number of DAases were reported before early 2000s. Advancements in whole-genome sequencing and searching tool of the biosynthetic gene clusters of the secondary metabolites facilitate the identification of plausible DAases. Thus, during past 5 years, nine DAases have been characterized by genetic and biochemical analyses. These include a detailed functional analysis of SpnF that solely catalyzes [4+2] cycloaddition, a structural analysis of spirotetramate-forming enzyme PyrI4 complexed with the corresponding cycloadduct, and DAases catalyzing decalin formation and macrocyclic pyridine formation. Together with decalin-forming enzymes and macrocyclic pyridine-forming enzymes, these results provided sufficient data to discuss catalytic mechanism of DAases and nature’s strategy for molecular diversification of linear chain intermediates derived from polyketide and ribosomal peptide biosynthetic machinery.
Co-reporter:Hideaki Oikawa and Yi Tang
The Journal of Antibiotics 2016 69(7) pp:471-472
Publication Date(Web):2016-07-01
DOI:10.1038/ja.2016.69
It is our great pleasure to be Guest Editors of this special issue of The Journal of Antibiotics dedicated to Professor David E Cane, upon his long-standing contribution as an editorial board member of this Journal. For over 45 years, Prof. Cane devoted much of his life to studying the biosynthesis of natural products.
Co-reporter:Ying Ye;Dr. Atsushi Minami;Yuya Igarashi;Dr. Miho Izumikawa;Dr. Myco Umemura;Dr. Nozomi Nagano;Dr. Masayuki Machida;Dr. Teppei Kawahara;Dr. Kazuo Shin-ya;Dr. Katsuya Gomi;Dr. Hideaki Oikawa
Angewandte Chemie 2016 Volume 128( Issue 28) pp:8204-8207
Publication Date(Web):
DOI:10.1002/ange.201602611
Abstract
The biosynthetic machinery of the first fungal ribosomally synthesized and post-translationally modified peptide (RiPP) ustiloxin B was elucidated through a series of gene inactivation and heterologous expression studies. The results confirmed an essential requirement for novel oxidases possessing the DUF3328 motif for macrocyclization, and highly unique side-chain modifications by three oxidases (UstCF1F2) and a pyridoxal 5′-phosphate (PLP)-dependent enzyme (UstD). These findings provide new insight into the expression of the RiPP gene clusters found in various fungi.
Co-reporter:Ying Ye;Dr. Atsushi Minami;Yuya Igarashi;Dr. Miho Izumikawa;Dr. Myco Umemura;Dr. Nozomi Nagano;Dr. Masayuki Machida;Dr. Teppei Kawahara;Dr. Kazuo Shin-ya;Dr. Katsuya Gomi;Dr. Hideaki Oikawa
Angewandte Chemie International Edition 2016 Volume 55( Issue 28) pp:8072-8075
Publication Date(Web):
DOI:10.1002/anie.201602611
Abstract
The biosynthetic machinery of the first fungal ribosomally synthesized and post-translationally modified peptide (RiPP) ustiloxin B was elucidated through a series of gene inactivation and heterologous expression studies. The results confirmed an essential requirement for novel oxidases possessing the DUF3328 motif for macrocyclization, and highly unique side-chain modifications by three oxidases (UstCF1F2) and a pyridoxal 5′-phosphate (PLP)-dependent enzyme (UstD). These findings provide new insight into the expression of the RiPP gene clusters found in various fungi.
Co-reporter:Ying Ye; Atsushi Minami; Attila Mandi; Chengwei Liu; Tohru Taniguchi; Tomohisa Kuzuyama; Kenji Monde; Katsuya Gomi
Journal of the American Chemical Society 2015 Volume 137(Issue 36) pp:11846-11853
Publication Date(Web):September 2, 2015
DOI:10.1021/jacs.5b08319
Genome mining is a promising method to discover novel secondary metabolites in the postgenomic era. We applied the Aspergillus oryzae heterologous expression system to functionally characterize cryptic bifunctional terpene synthase genes found in fungal genomes and identified the sesterfisherol synthase gene (NfSS) from Neosartorya fischeri. Sesterfisherol contains a characteristic 5–6–8–5 tetracyclic ring system and is modified by cytochrome P450 monooxygenase (NfP450) to sesterfisheric acid. The cyclization mechanism was proposed on the basis of the analysis of in vivo and in vitro enzymatic reactions with isotopically labeled precursors. The mechanism involves C1 cation–olefin IV–olefin V cyclization followed by five hydride shifts, allowing us to propose a unified biogenesis for sesterterpenes branching from bicyclic (5–15), tricyclic (5–12–5), and tetracyclic (5–6–8–5) cation intermediates. Furthermore, the mechanism is distinct from that of a separate class of di/sesterterpenes including fusicoccins and ophiobolins. The difference between mechanisms is consistent with phylogenetic analysis of bifunctional terpene synthases, suggesting that the amino acid sequence reflects the initial cyclization mode, which is most likely related to the initial conformation of a linear prenyl diphosphate.
Co-reporter:Takahiro Ugai, Atsushi Minami, Ryuya Fujii, Mizuki Tanaka, Hiroki Oguri, Katsuya Gomi and Hideaki Oikawa
Chemical Communications 2015 vol. 51(Issue 10) pp:1878-1881
Publication Date(Web):15 Dec 2014
DOI:10.1039/C4CC09512J
A unique highly reducing polyketide synthase (HR-PKS) with a reductase domain was identified in a betaenone biosynthetic gene cluster. Successful heterologous expression and characterization of the HR-PKS and trans-acting enoyl reductase (ER) provide insights into the core structure formation with a decalin scaffold and allow reconstitution of the betaenone biosynthetic machinery.
Co-reporter:Ryuya Fujii, Yusuke Matsu, Atsushi Minami, Shota Nagamine, Ichiro Takeuchi, Katsuya Gomi, and Hideaki Oikawa
Organic Letters 2015 Volume 17(Issue 22) pp:5658-5661
Publication Date(Web):November 11, 2015
DOI:10.1021/acs.orglett.5b02934
To elucidate the general biosynthetic pathway of fungal dimeric anhydrides, a gene cluster for the biosynthesis of the antihy-percholesterolemic agent phomoidride was identified by heterologous expression of candidate genes encoding the highly reducing polyketide synthase, alkylcitrate synthase (ACS), and alkylcitrate dehydratase (ACDH). An in vitro analysis of ACS and ACDH revealed that they give rise to anhydride monomers. Based on the established monomer biosynthesis, we propose a general biogenesis of dimeric anhydrides involving a single donor unit and four acceptor units.
Co-reporter:Dr. Chengwei Liu;Koichi Tagami;Dr. Atsushi Minami;Tomoyuki Matsumoto;Jens Christian Frisvad;Hideyuki Suzuki;Jun Ishikawa;Katsuya Gomi;Dr. Hideaki Oikawa
Angewandte Chemie 2015 Volume 127( Issue 19) pp:
Publication Date(Web):
DOI:10.1002/ange.201502797
Co-reporter:Dr. Chengwei Liu;Koichi Tagami;Dr. Atsushi Minami;Tomoyuki Matsumoto;Jens Christian Frisvad;Hideyuki Suzuki;Jun Ishikawa;Katsuya Gomi;Dr. Hideaki Oikawa
Angewandte Chemie International Edition 2015 Volume 54( Issue 19) pp:5748-5752
Publication Date(Web):
DOI:10.1002/anie.201501072
Abstract
Penitrem A is one of the most elaborated members of the fungal indole diterpenes. Two separate penitrem gene clusters were identified using genomic and RNA sequencing data, and 13 out of 17 transformations in the penitrem biosynthesis were elucidated by heterologous reconstitution of the relevant genes. These reactions involve 1) a prenylation-initiated cationic cyclization to install the bicyclo[3.2.0]heptane skeleton (PtmE), 2) a two-step P450-catalyzed oxidative processes forming the unique tricyclic penitrem skeleton (PtmK and PtmU), and 3) five sequential oxidative transformations (PtmKULNJ). Importantly, without conventional gene disruption, reconstitution of the biosynthetic machinery provided sufficient data to determine the pathway. It was thus demonstrated that the Aspergillus oryzae reconstitution system is a powerful method for studying the biosynthesis of complex natural products.
Co-reporter:Dr. Chengwei Liu;Koichi Tagami;Dr. Atsushi Minami;Tomoyuki Matsumoto;Jens Christian Frisvad;Hideyuki Suzuki;Jun Ishikawa;Katsuya Gomi;Dr. Hideaki Oikawa
Angewandte Chemie International Edition 2015 Volume 54( Issue 19) pp:
Publication Date(Web):
DOI:10.1002/anie.201502797
Co-reporter:Dr. Chengwei Liu;Koichi Tagami;Dr. Atsushi Minami;Tomoyuki Matsumoto;Jens Christian Frisvad;Hideyuki Suzuki;Jun Ishikawa;Katsuya Gomi;Dr. Hideaki Oikawa
Angewandte Chemie 2015 Volume 127( Issue 19) pp:5840-5844
Publication Date(Web):
DOI:10.1002/ange.201501072
Abstract
Penitrem A is one of the most elaborated members of the fungal indole diterpenes. Two separate penitrem gene clusters were identified using genomic and RNA sequencing data, and 13 out of 17 transformations in the penitrem biosynthesis were elucidated by heterologous reconstitution of the relevant genes. These reactions involve 1) a prenylation-initiated cationic cyclization to install the bicyclo[3.2.0]heptane skeleton (PtmE), 2) a two-step P450-catalyzed oxidative processes forming the unique tricyclic penitrem skeleton (PtmK and PtmU), and 3) five sequential oxidative transformations (PtmKULNJ). Importantly, without conventional gene disruption, reconstitution of the biosynthetic machinery provided sufficient data to determine the pathway. It was thus demonstrated that the Aspergillus oryzae reconstitution system is a powerful method for studying the biosynthesis of complex natural products.
Co-reporter:Atsushi Minami, Toyoyuki Ose, Kyohei Sato, Azusa Oikawa, Kimiko Kuroki, Katsumi Maenaka, Hiroki Oguri, and Hideaki Oikawa
ACS Chemical Biology 2014 Volume 9(Issue 2) pp:562
Publication Date(Web):December 9, 2013
DOI:10.1021/cb4006485
Multistep catalysis of epoxide hydrolase/cyclase in the epoxide opening cascade is an intriguing issue in polyether biosynthesis. A pair of structurally homologous epoxide hydrolases was found in gene clusters of ionophore polyethers. In the epoxide opening reactions with MonBI and MonBII involved in monensin biosynthesis, we found that MonBII and catalytically inactive MonBI mutant catalyzed two-step reactions of bisepoxide substrate analogue to afford bicyclic product although MonBII alone catalyzed only the first cyclization. The X-ray crystal structure of MonBI dimers suggested the importance of the KSD motif in MonBI/MonBI interaction, which was further supported by gel filtration chromatography of wild-type MonBI and mutant MonBI. The involvement of the KSD motif in heterodimer formation was confirmed by in vitro assay. Direct evidence of MonBI/MonBII interaction was obtained by native mass spectrometry. Its dissociation constant was determined as 2.21 × 10–5 M by surface plasmon resonance. Our results suggested the involvement of an allosteric regulation mechanism by MonBI/MonBII interaction in monensin skeletal construction.
Co-reporter:Dr. Tomoshige Hiratsuka;Hideaki Suzuki;Ryo Kariya;Takashi Seo;Dr. Atsushi Minami ;Dr. Hideaki Oikawa
Angewandte Chemie International Edition 2014 Volume 53( Issue 21) pp:5423-5426
Publication Date(Web):
DOI:10.1002/anie.201402623
Abstract
The biosynthetic gene cluster of antifungal agent jawsamycin (FR-900848) has been identified by heterologous expression. A series of gene inactivations and in vitro and in vivo analysis of key enzymes in the biosynthetic pathway established their functions. A novel mechanism involving a radical S-adenosyl methionine (SAM) cyclopropanase collaborating with an iterative polyketide synthase is proposed for the construction of the unique polycyclopropanated backbone. Our reconstitution system sets the stage for studying the catalytic mechanism of this intriguing contiguous cyclopropanation.
Co-reporter:Koichi Tagami;Dr. Atsushi Minami;Ryuya Fujii;Dr. Chengwei Liu;Dr. Mizuki Tanaka; Dr. Katsuya Gomi; Dr. Tohru Dairi; Dr. Hideaki Oikawa
ChemBioChem 2014 Volume 15( Issue 14) pp:2076-2080
Publication Date(Web):
DOI:10.1002/cbic.201402195
Abstract
Reconstitution of the biosynthetic machinery for fungal secondary metabolites in Aspergillus oryzae provides an opportunity both for stepwise determination of the biosynthetic pathways and the total biosynthesis of fungal natural products. However, to maximize the utility of the reconstitution system, a simple and rapid strategy for the introduction of heterologous genes into A. oryzae is required. In this study, we demonstrated an effective method for introducing multiple genes involved in the biosynthesis of fungal metabolites by using the expression vectors pUARA2 and pUSA2, each of which contains two cloning sites. The successful introduction of all the aflatrem biosynthetic genes (seven genes in total) after two rounds of transformation enabled the total biosynthesis of aflatrem. This rapid reconstitution strategy will facilitate the functional analysis of the biosynthetic machinery of fungal metabolites.
Co-reporter:Dr. Tomoshige Hiratsuka;Hideaki Suzuki;Ryo Kariya;Takashi Seo;Dr. Atsushi Minami ;Dr. Hideaki Oikawa
Angewandte Chemie 2014 Volume 126( Issue 21) pp:5527-5530
Publication Date(Web):
DOI:10.1002/ange.201402623
Abstract
The biosynthetic gene cluster of antifungal agent jawsamycin (FR-900848) has been identified by heterologous expression. A series of gene inactivations and in vitro and in vivo analysis of key enzymes in the biosynthetic pathway established their functions. A novel mechanism involving a radical S-adenosyl methionine (SAM) cyclopropanase collaborating with an iterative polyketide synthase is proposed for the construction of the unique polycyclopropanated backbone. Our reconstitution system sets the stage for studying the catalytic mechanism of this intriguing contiguous cyclopropanation.
Co-reporter:Koichi Tagami ; Chengwei Liu ; Atsushi Minami ; Motoyoshi Noike ; Tetsuya Isaka ; Shuhei Fueki ; Yoshihiro Shichijo ; Hiroaki Toshima ; Katsuya Gomi ; Tohru Dairi
Journal of the American Chemical Society 2013 Volume 135(Issue 4) pp:1260-1263
Publication Date(Web):January 11, 2013
DOI:10.1021/ja3116636
Indole-diterpenes represented by paxilline share a common pentacyclic core skeleton derived from indole and geranylgeranyl diphosphate. To shed light on the detailed biosynthetic mechanism of the paspaline-type hexacyclic skeleton, we examined the reconstitution of paxilline biosynthetic machinery in Aspergillus oryzae NSAR1. Stepwise introduction of the six pax genes enabled us to isolate all biosynthetic intermediates and to synthesize paxilline. In vitro and in vivo studies on the key enzymes, prenyltransferase PaxC and cyclase PaxB, allowed us to elucidate actual substrates of these enzymes. Using the isolated and the synthesized epoxide substrates, the highly intriguing stepwide epoxidation/cyclization mechanism for the construction of core structure has been confirmed. In addition, we also demonstrated “tandem transformation” to simultaneously introduce two genes using a single vector (paxG/paxB, pAdeA; paxP/paxQ, pUNA). This may provide further option for the reconstitution strategy to synthesize more complex fungal metabolites.
Co-reporter:Atsushi Minami, Hiroki Oguri, Kenji Watanabe, Hideaki Oikawa
Current Opinion in Chemical Biology 2013 Volume 17(Issue 4) pp:555-561
Publication Date(Web):August 2013
DOI:10.1016/j.cbpa.2013.06.004
•Enzymatic polyether assembly mechanism in ionophore antibiotic lasalocid.•Anti-Baldwin cyclic ether formation catalyzed by polyether biosynthetic enzyme Lsd19.•Crystal structure of Lsd19 catalyzing polyether epoxide opening cascade.Diversity of natural polycyclic polyethers originated from very simple yet versatile strategy consisting of epoxidation of linear polyene followed by epoxide opening cascade. To understand two-step enzymatic transformations at molecular basis, a flavin containing monooxygenase (EPX) Lsd18 and an epoxide hydrolase (EH) Lsd19 were selected as model enzymes for extensive investigation on substrate specificity, catalytic mechanism, cofactor requirement and crystal structure. This pioneering study on prototypical lasalocid EPX and EH provides insight into detailed mechanism of ionophore polyether assembly machinery and clarified remaining issues for polyether biosynthesis.
Co-reporter:Ryuya Fujii, Atsushi Minami, Katsuya Gomi, Hideaki Oikawa
Tetrahedron Letters 2013 Volume 54(Issue 23) pp:2999-3002
Publication Date(Web):5 June 2013
DOI:10.1016/j.tetlet.2013.03.120
Cytochalasins are an important class of fungal natural products in view of structural diversity and biological activities. Although their biosynthetic studies have been examined extensively, the detailed molecular assembly mechanism remains to be solved. We have succeeded to heterologously express the cytochalasin polyketide synthase–non-ribosomal peptide synthetase (PKS–NRPS) hybrid gene ccsA and the trans-acting enoyl-CoA reductase gene ccsC in Aspergillus oryzae. The resultant transformant produced a novel metabolite possessing the cytochalasin backbone. This established that CcsA is capable of constructing the octaketide connected with phenylalanine in collaboration with CcsC, and that CcsA R domain catalyzes reductive cleavage of the thio-tethered PKS–NRPS product.Aspergillus oryzae NSAR1.
Co-reporter:Atsushi Minami ; Mayu Shimaya ; Gaku Suzuki ; Akira Migita ; Sandip S. Shinde ; Kyohei Sato ; Kenji Watanabe ; Tomohiro Tamura ; Hiroki Oguri
Journal of the American Chemical Society 2012 Volume 134(Issue 17) pp:7246-7249
Publication Date(Web):April 17, 2012
DOI:10.1021/ja301386g
Enantioselective epoxidation followed by regioselective epoxide opening reaction are the key processes in construction of the polyether skeleton. Recent genetic analysis of ionophore polyether biosynthetic gene clusters suggested that flavin-containing monooxygenases (FMOs) could be involved in the oxidation steps. In vivo and in vitro analyses of Lsd18, an FMO involved in the biosynthesis of polyether lasalocid, using simple olefin or truncated diene of a putative substrate as substrate mimics demonstrated that enantioselective epoxidation affords natural type mono- or bis-epoxide in a stepwise manner. These findings allow us to figure out enzymatic polyether construction in lasalocid biosynthesis.
Co-reporter:Kento Koketsu, Atsushi Minami, Kenji Watanabe, Hiroki Oguri, Hideaki Oikawa
Current Opinion in Chemical Biology 2012 Volume 16(1–2) pp:142-149
Publication Date(Web):April 2012
DOI:10.1016/j.cbpa.2012.02.021
Nonribosomal peptide synthetase (NRPS) is a programmable modular machinery that produces a number of biologically active small-molecule peptides. Saframycin A is a potent antitumor antibiotic with a unique pentacyclic tetrahydroisoquinoline scaffold. We found that the nonribosomal peptide synthetase SfmC catalyzes a seven-step transformation of readily synthesized dipeptidyl substrates with long acyl chains into a complex saframycin scaffold. Based on a series of enzymatic reactions, we proposed a detailed mechanism involving the reduction of various peptidyl thioesters by a single R domain followed by iterative C domain-mediated Pictet-Spengler reactions. This shows that NRPSs possess a remarkable capability to acquire novel function for diversifying structures of peptide natural products.Highlights► Remarkable multiple-catalysis of NRPS: construction of complex saframycin scaffold. ► Novel functions of nonribosomal peptide synthetase found in saframycin biosynthesis. ► Pictet-Spenglerase involved in tetrahydroisoquinoline antibiotic biosynthesis.
Co-reporter:Atsushi Minami, Akira Migita, Daiki Inada, Kinya Hotta, Kenji Watanabe, Hiroki Oguri, and Hideaki Oikawa
Organic Letters 2011 Volume 13(Issue 7) pp:1638-1641
Publication Date(Web):March 4, 2011
DOI:10.1021/ol200100e
Our recent findings of the first epoxide hydrolase Lsd19, involved in lasalocid A biosynthesis, led us to investigate a long-standing controversial issue on the mechanism of enzymatic epoxide-opening cascades. The site-directed mutagenesis and domain dissection analysis to reveal the mechanism of the reaction catalyzed by Lsd19 is examined, especially in the role of acidic amino acid pair and catalytic domains.
Co-reporter:Kyohei Sato, Atsushi Minami, Toyoyuki Ose, Hiroki Oguri, Hideaki Oikawa
Tetrahedron Letters 2011 Volume 52(Issue 41) pp:5277-5280
Publication Date(Web):12 October 2011
DOI:10.1016/j.tetlet.2011.07.145
Enzymatic epoxide-opening cascade is one of the key biosynthetic processes for constructing structurally diverse polyethers. Here we report the first in vitro analysis of the cyclization catalyzed by two epoxide hydrolases, MonBI and MonBII involved in monensin biosynthesis, using simple epoxy-alcohols and the unprecedented synergistic effect on the epoxide-opening activity between these epoxide hydrolases.
Co-reporter:Yuki Hirose, Kenji Watanabe, Atsushi Minami, Takemichi Nakamura, Hiroki Oguri and Hideaki Oikawa
The Journal of Antibiotics 2011 64(1) pp:117-122
Publication Date(Web):November 24, 2010
DOI:10.1038/ja.2010.142
Quinomycin antibiotics, represented by echinomycin, are an important class of antitumor antibiotics. We have recently succeeded in identification of biosynthetic gene clusters of echinomycin and SW-163D, and have achieved heterologous production of echinomycin in Escherichia coli. In addition, we have engineered echinomycin non-ribosomal peptide synthetase to generate echinomycin derivatives. However, the biosynthetic pathways of intercalative chromophores quinoxaline-2-carboxylic acid (QXC) and 3-hydroxyquinaldic acid (HQA), which are important for biological activity, were not fully elucidated. Here, we report experiments involving incorporation of a putative advanced precursor, (2S, 3R)-[6′-2H]-3-hydroxy-L-kynurenine, and functional analysis of the enzymes Swb1 and Swb2 responsible for late-stage biosynthesis of HQA. On the basis of these experimental results, we propose biosynthetic pathways for both QXC and HQA through the common intermediate 3-hydroxy-L-kynurenine.
Co-reporter:Yusuke Matsuura, Yoshihiro Shichijo, Atsushi Minami, Akira Migita, Hiroki Oguri, Mami Watanabe, Tetsuo Tokiwano, Kenji Watanabe and Hideaki Oikawa
Organic Letters 2010 Volume 12(Issue 10) pp:2226-2229
Publication Date(Web):April 15, 2010
DOI:10.1021/ol100541e
Recently, we reported that the epoxide hydrolase Lsd19, the first enzyme shown to catalyze epoxide-opening cascades, can catalyze the conversion of a putative bisepoxide intermediate to polyether antibiotic lasalocid, which involves energetically disfavored 6-endo-tet cyclization of the epoxy alcohol. Here, we examined the substrate tolerance of Lsd19. Lsd19 accepts various substrate analogues differing in the left segment of lasalocid and epoxide stereochemistry to afford either THF−THP or THF−THF products with excellent regioselectivity.
Co-reporter:Kenji Watanabe ; Kinya Hotta ; Mino Nakaya ; Alex P. Praseuth ; Clay C. C. Wang ; Daiki Inada ; Kosaku Takahashi ; Eri Fukushi ; Hiroki Oguri
Journal of the American Chemical Society 2009 Volume 131(Issue 26) pp:9347-9353
Publication Date(Web):June 10, 2009
DOI:10.1021/ja902261a
Natural products display impressive activities against a wide range of targets, including viruses, microbes, and tumors. However, their clinical use is hampered frequently by their scarcity and undesirable toxicity. Not only can engineering Escherichia coli for plasmid-based pharmacophore biosynthesis offer alternative means of simple and easily scalable production of valuable yet hard-to-obtain compounds, but also carries a potential for providing a straightforward and efficient means of preparing natural product analogs. The quinomycin family of nonribosomal peptides, including echinomycin, triostin A, and SW-163s, are important secondary metabolites imparting antibiotic antitumor activity via DNA bisintercalation. Previously we have shown the production of echinomycin and triostin A in E. coli using our convenient and modular plasmid system to introduce these heterologous biosynthetic pathways into E. coli. However, we have yet to develop a novel biosynthetic pathway capable of producing bioactive unnatural natural products in E. coli. Here we report an identification of a new gene cluster responsible for the biosynthesis of SW-163s that involves previously unknown biosynthesis of (+)-(1S, 2S)-norcoronamic acid and generation of aliphatic side chains of various sizes via iterative methylation of an unactivated carbon center. Substituting an echinomycin biosynthetic gene with a gene from the newly identified SW-163 biosynthetic gene cluster, we were able to rationally re-engineer the plasmid-based echinomycin biosynthetic pathway for the production of a novel bioactive compound in E. coli.
Co-reporter:Tetsuo Tokiwano, Hiroaki Watanabe, Takashi Seo and Hideaki Oikawa
Chemical Communications 2008 (Issue 45) pp:6016-6018
Publication Date(Web):14 Oct 2008
DOI:10.1039/B809610D
We were able to show the predominant incorporation of a single enantiomer and intact incorporation of multiply labelled synthetic diketide precursors (14 and 16), which established the intermediacy of cyclopropanated diketide and led to our proposal for the unprecedented biological cyclopropanation, viaPKS (polyketide synthase) having a novel cyclopropanase domain, in the biosynthesis of FR-900848 (1).
Co-reporter:Kento Koketsu, Hiroki Oguri, Kenji Watanabe, Hideaki Oikawa
Chemistry & Biology 2008 Volume 15(Issue 8) pp:818-828
Publication Date(Web):25 August 2008
DOI:10.1016/j.chembiol.2008.05.022
Excised thioesterase domains are versatile catalysts for macrocyclization. However, thioesterase-catalyzed cyclization is often precluded due to the occurrence of hydrolysis and product inhibition. To circumvent these obstacles, we devised an unprecedented strategy: coincubation with DNA to capture the cyclic products possessing DNA-binding properties. In experiments involving echinomycin thioesterase-catalyzed macrolactonization leading to the cyclic triostin A analog TANDEM, we found that the addition of DNA drastically improved the yield of TANDEM (19% → 67%), with a complete reversal of the cyclization:hydrolysis ratio (1:2 → 18:1). Furthermore, the applicability of this protocol was demonstrated for a variety of substrates. The results described herein provide insight into the mechanism of echinomycin thioesterase-catalyzed conversions and also pave the way for chemoenzymatic synthesis of the quinoxaline antibiotics and their analogs.
Co-reporter:Kenji Watanabe and Hideaki Oikawa
Organic & Biomolecular Chemistry 2007 vol. 5(Issue 4) pp:593-602
Publication Date(Web):21 Dec 2006
DOI:10.1039/B615589H
During the past decade, numerous gene clusters responsible for the biosynthesis of important natural products have been identified from a variety of organisms. Heterologous expression utilizing E. coli has been employed to provide proteins for mechanistic understanding and structural analyses. It was very recently shown that this system is also capable of de novo production of biologically active forms of heterologous nonribosomal peptides, echinomycin and triostin A, through the introduction of genes encoding modules responsible for their assembly into this model bacterial host. The superlative advantage of using E. coli as a heterologous host is the availability of a wealth of well-established molecular biological techniques for its genetic and metabolic manipulation. The platform described above which was developed in our laboratory is ideal for use in the production of metabolites found in marine and symbiotic bacteria that are not amenable to artificial cultivation. Development and tailoring of our system will allow for the design of these natural products and ultimately combinatorial yet rational modification of these compounds. This review focuses on the heterologous expression of biosynthetic gene clusters for the assembly of therapeutically potent compounds.
Co-reporter:Hiroaki Watanabe, Tetsuo Tokiwano and Hideaki Oikawa
The Journal of Antibiotics 2006 59(9) pp:607-610
Publication Date(Web):
DOI:10.1038/ja.2006.82
Biosynthetic studies of the antifungal agent, FR-900848, were undertaken by feeding experiments with D-[U-13C6]glucose, L-[4-13C]aspartate, [5,5-2H2]dihydrouridine and [5,5-2H2]dihydrouracil. The 5"-amino-5"-deoxy-5',6'-dihydrouridine moiety was derived from ribose and aspartate. Based on the feeding experiments, a detailed biosynthetic pathway producing the aminodeoxydihydrouridine moiety of FR-900848 was proposed.
Co-reporter:Tetsuo Tokiwano, Taeko Endo, Tae Tsukagoshi, Hitoshi Goto, Eri Fukushi and Hideaki Oikawa
Organic & Biomolecular Chemistry 2005 vol. 3(Issue 15) pp:2713-2722
Publication Date(Web):21 Jun 2005
DOI:10.1039/B506411B
To obtain insight into how the cyclization pathway is controlled, the mechanism of diterpene synthase reactions (the putative phomactatriene synthase and taxadiene synthases) involving the same intermediate was investigated in detail. The mechanism of the initial transformation of GGDP to verticillen-12-yl cation (A+) was proposed based on the labelling pattern of phomactatriene (9a) obtained in the feeding experiments with 13C-labelled acetates. To obtain information on the reaction pathway of A+ to 9a and taxadiene (10), reactions of verticillol (5) with various acids were conducted. Structural determination of products 9b, 9c, 13a, 13b and 14 allowed us to propose a reaction pathway via cations A+, D+, E+, F+ and G+. Identification of hydrocarbons 6, 9b, 13a and 13b in mycelial extracts of phomactin-producing fungus supported the proposed reaction mechanism. Based on the results of ab initio calculations for highly flexible cation intermediates, a mechanism is proposed.
Co-reporter:Yuta Fujita, Hiroki Oguri and Hideaki Oikawa
The Journal of Antibiotics 2005 58(6) pp:425-427
Publication Date(Web):
DOI:10.1038/ja.2005.56
A potentially general approach for elucidating the absolute configuration of N-hydroxypyridone antibiotics has been developed. One member of this family of antibiotics, PF1140, was efficiently purified from a crude fungal extract following allylation of its N-hydroxyl group. Removal of the resultant allyl group permitted regeneration of the N-hydroxyl group as well as conversion into the corresponding pyridone derivative. The stereochemistry of PF1140 including the absolute configuration was established by X-ray crystallographic analysis of the S-2-methoxy-2-(1-naphthyl)propionic ester derivative.
Co-reporter:Tetsuo Tokiwano, Eri Fukushi, Taeko Endo and Hideaki Oikawa
Chemical Communications 2004 (Issue 11) pp:1324-1325
Publication Date(Web):10 May 2004
DOI:10.1039/B401377H
The stereochemical course of GGDP cyclization in the biosynthesis of phomactins is proposed by the stereochemistry of a cyclization product, phomacta-1(14),3,7-triene, isolated from Phomasp. and the results of incorporation experiments with [1-13C]- and [1,2-13C2]acetates.
Co-reporter:Tomoshige Hiratsuka, Kento Koketsu, Atsushi Minami, Shunsuke Kaneko, Chiaki Yamazaki, Kenji Watanabe, Hiroki Oguri, Hideaki Oikawa
Chemistry & Biology (19 December 2013) Volume 20(Issue 12) pp:
Publication Date(Web):19 December 2013
DOI:10.1016/j.chembiol.2013.10.011
•Identification of common genes for core assembly of quinocarcin and SF-1739•Unique building unit dehydroarginine is produced by α-KG-dependent dioxygenase•A complex of NRPSs and MbtH is proposed to catalyze multistep core assembly•MbtH protein affects both expression level and activity of quinocarcin NRPSsQuinocarcin and SF-1739, potent antitumor antibiotics, share a common tetracyclic tetrahydroisoquinoline (THIQ)-pyrrolidine core scaffold. Herein, we describe the identification of their biosynthetic gene clusters and biochemical analysis of Qcn18/Cya18 generating the previously unidentified extender unit dehydroarginine, which is a component of the pyrrolidine ring. ATP-inorganic pyrophosphate exchange experiments with five nonribosomal peptide synthetases (NRPSs) enabled us to identify their substrates. On the basis of these data, we propose that a biosynthetic pathway comprising a three-component NRPS/MbtH family protein complex, Qcn16/17/19, plays a key role in the construction of tetracyclic THIQ-pyrrolidine core scaffold involving sequential Pictet-Spengler and intramolecular Mannich reactions. Furthermore, data derived from gene inactivation experiments led us to propose late-modification steps of quinocarcin.Figure optionsDownload full-size imageDownload high-quality image (165 K)Download as PowerPoint slide
Co-reporter:Tomoshige Hiratsuka, Hideaki Suzuki, Atsushi Minami and Hideaki Oikawa
Organic & Biomolecular Chemistry 2017 - vol. 15(Issue 5) pp:NaN1079-1079
Publication Date(Web):2016/12/22
DOI:10.1039/C6OB02675C
Jawsamycin is a polyketide–nucleoside hybrid with a unique polycyclopropane moiety on a single polyketide chain. The unexpected isolation of cyclopropane deficient jawsamycin analogs allowed us to propose a stepwise cyclopropanation mechanism for the enzymatic synthesis of this polyketide. The concise timing of the cyclopropanation could be regulated by a delicate balance between reaction rates of the condensation and cyclopropanation reactions.
Co-reporter:Kenji Watanabe and Hideaki Oikawa
Organic & Biomolecular Chemistry 2007 - vol. 5(Issue 4) pp:NaN602-602
Publication Date(Web):2006/12/21
DOI:10.1039/B615589H
During the past decade, numerous gene clusters responsible for the biosynthesis of important natural products have been identified from a variety of organisms. Heterologous expression utilizing E. coli has been employed to provide proteins for mechanistic understanding and structural analyses. It was very recently shown that this system is also capable of de novo production of biologically active forms of heterologous nonribosomal peptides, echinomycin and triostin A, through the introduction of genes encoding modules responsible for their assembly into this model bacterial host. The superlative advantage of using E. coli as a heterologous host is the availability of a wealth of well-established molecular biological techniques for its genetic and metabolic manipulation. The platform described above which was developed in our laboratory is ideal for use in the production of metabolites found in marine and symbiotic bacteria that are not amenable to artificial cultivation. Development and tailoring of our system will allow for the design of these natural products and ultimately combinatorial yet rational modification of these compounds. This review focuses on the heterologous expression of biosynthetic gene clusters for the assembly of therapeutically potent compounds.
Co-reporter:Takahiro Ugai, Atsushi Minami, Ryuya Fujii, Mizuki Tanaka, Hiroki Oguri, Katsuya Gomi and Hideaki Oikawa
Chemical Communications 2015 - vol. 51(Issue 10) pp:NaN1881-1881
Publication Date(Web):2014/12/15
DOI:10.1039/C4CC09512J
A unique highly reducing polyketide synthase (HR-PKS) with a reductase domain was identified in a betaenone biosynthetic gene cluster. Successful heterologous expression and characterization of the HR-PKS and trans-acting enoyl reductase (ER) provide insights into the core structure formation with a decalin scaffold and allow reconstitution of the betaenone biosynthetic machinery.
Co-reporter:Tetsuo Tokiwano, Hiroaki Watanabe, Takashi Seo and Hideaki Oikawa
Chemical Communications 2008(Issue 45) pp:NaN6018-6018
Publication Date(Web):2008/10/14
DOI:10.1039/B809610D
We were able to show the predominant incorporation of a single enantiomer and intact incorporation of multiply labelled synthetic diketide precursors (14 and 16), which established the intermediacy of cyclopropanated diketide and led to our proposal for the unprecedented biological cyclopropanation, viaPKS (polyketide synthase) having a novel cyclopropanase domain, in the biosynthesis of FR-900848 (1).
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![2(1H)-Pyridinone,5-(2,5-dihydroxy-7-oxabicyclo[4.1.0]hept-2-yl)-1,4-dihydroxy-3-[(1,2,4a,5,6,7,8,8a-octahydro-2-methyl-1-naphthalenyl)carbonyl]-(9CI)](http://img.cochemist.com/ccimg/134900/134822-63-2.png)
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![Cyclopenta[4,5]cyclooct[1,2-f]indene-6-carboxaldehyde,1,2,3,3a,4,6a,7,7a,8,9,10,10a,11,11a-tetradecahydro-3,7a,12-trimethyl-10-(1-methylethyl)-2-(b-D-xylopyranosyloxy)-, (2S,3S,3aS,6aS,7aR,10S,10aS,11aS)-(9CI)](http://img.cochemist.com/ccimg/122600/122535-46-0.png)
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![1(2H)-Naphthalenone, octahydro-2,7-dihydroxy-4-[(2Z)-3-hydroxy-1-oxo-2-propen-1-yl]-2,4,5,7-tetramethyl-3-[(1R)-1-methylpropyl]-,](/data/chemimg/1992600/85269-25-6.png)
![1(2H)-Naphthalenone, octahydro-2,7-dihydroxy-4-[(2Z)-3-hydroxy-1-oxo-2-propen-1-yl]-2,4,5,7-tetramethyl-3-[(1R)-1-methylpropyl]-,](/data/chemimg/1992600/85269-25-6_b.png)
![(2S,3R,4R,4aS,5R,7R,8aS)-3-[(2R)-butan-2-yl]-2,7-dihydroxy-4-(3-hydroxypropanoyl)-2,4,5,7-tetramethyloctahydronaphthalen-1(2H)-one](http://img.cochemist.com/ccimg/85300/85269-23-4.png)
![(2S,3R,4R,4aS,5R,7R,8aS)-3-[(2R)-butan-2-yl]-2,7-dihydroxy-4-(3-hydroxypropanoyl)-2,4,5,7-tetramethyloctahydronaphthalen-1(2H)-one](http://img.cochemist.com/ccimg/85300/85269-23-4_b.png)
![(2R,3S,3aR,4aS,4bS,6aR,7S,7dR,8S,9aR,14bS,14cR,16aS)-14b,14c,17,17-tetramethyl-2-(1-methylethenyl)-10-methylidene-3,3a,6,6a,7,8,9,9a,10,11,14,14b,14c,15,16,16a-hexadecahydro-2H,4bH-7,8-(epoxymethano)cyclobuta[5,6]benzo[1,2-e]oxireno[4',4a']chromeno[5',6':](http://img.cochemist.com/ccimg/78300/78213-66-8.png)
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![(2R,3R,4bS,6aR,7S,7dS,8S,9aS,14bS,14cR,16aS)-14b,14c,17,17-tetramethyl-2-(1-methylethenyl)-10-methylidene-2,3,5,6,6a,7,7d,8,9,9a,10,11,14,14b,14c,15,16,16a-octadecahydro-4bH-7,8-(epoxymethano)chromeno[5',6':6,7]indeno[1,2-b]cyclobuta[5,6]benzo[1,2-e]indol](http://img.cochemist.com/ccimg/78300/78213-64-6.png)
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![5b-hydroxy-2,2,13b,13c-tetramethyl-2,3,5b,6,7,7a,8,13,13b,13c,14,15-dodecahydro-4H-3,15a-epoxy[1]benzoxepino[6',7':6,7]indeno[1,2-b]indol-4-one](http://img.cochemist.com/ccimg/63800/63722-91-8.png)
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![7,8-(Epoxymethano)-4bH-1-benzopyrano[5',6':6,7]indeno[1,2-b]cyclobuta[5,6]benz[1,2-e]indole-3,4b-diol,12-chloro-2,3,5,6,6a,7,7d,8,9,9a,10,11,14,14b,14c,15,16,16a-octadecahydro-14b,14c,17,17-tetramethyl-10-methylene-2-(1-methylethenyl)-,(2R,3R,4bR,6aR,7S,7dS,8S,9aS,14bS,14cR,16aS)- (9CI)](http://img.cochemist.com/ccimg/37400/37318-84-6.png)
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![(3AR,4R,5R,6AS)-4-FORMYL-2-OXOHEXAHYDRO-2H-CYCLOPENTA[B]FURAN-5-Y<WBR />L 4-BIPHENYLCARBOXYLATE](http://img.cochemist.com/ccimg/100/72-89-9_b.png)
![[10b,10'b-Bi-10bH-pyrazino[1',2':1,5]pyrrolo[2,3-b]indole]-1,1',4,4'-tetrone,2,2',3,3',5a,5'a,6,6',11,11',11a,11'a-dodecahydro-3,3'-bis(phenylmethyl)-,(3S,3'S,5aR,5'aR,10bR,10'bR,11aS,11'aS)-](http://img.cochemist.com/ccimg/150900/150881-27-9.png)
![[10b,10'b-Bi-10bH-pyrazino[1',2':1,5]pyrrolo[2,3-b]indole]-1,1',4,4'-tetrone,2,2',3,3',5a,5'a,6,6',11,11',11a,11'a-dodecahydro-3,3'-bis(phenylmethyl)-,(3S,3'S,5aR,5'aR,10bR,10'bR,11aS,11'aS)-](http://img.cochemist.com/ccimg/150900/150881-27-9_b.png)
![Uridine,5'-deoxy-5,6-dihydro-5'-[[(2E,4E)-5-[(1R,1'R,1''R,1'''R,2S,2'R,2''R,2'''S)-2'''-[(1E)-2-[(1R,2R)-2-methylcyclopropyl]ethenyl][1,1':2',1'':2'',1'''-quatercyclopropan]-2-yl]-1-oxo-2,4-pentadien-1-yl]amino]-](http://img.cochemist.com/ccimg/120600/120500-69-8.png)
![Uridine,5'-deoxy-5,6-dihydro-5'-[[(2E,4E)-5-[(1R,1'R,1''R,1'''R,2S,2'R,2''R,2'''S)-2'''-[(1E)-2-[(1R,2R)-2-methylcyclopropyl]ethenyl][1,1':2',1'':2'',1'''-quatercyclopropan]-2-yl]-1-oxo-2,4-pentadien-1-yl]amino]-](http://img.cochemist.com/ccimg/120600/120500-69-8_b.png)
![5b-hydroxy-2,2,13b,13c-tetramethyl-9-(2-methylbut-3-en-2-yl)-2,3,5b,6,7,7a,8,13,13b,13c,14,15-dodecahydro-4H-3,15a-epoxy[1]benzoxepino[6',7':6,7]indeno[1,2-b]indol-4-one](http://img.cochemist.com/ccimg/70600/70553-75-2.png)
![5b-hydroxy-2,2,13b,13c-tetramethyl-9-(2-methylbut-3-en-2-yl)-2,3,5b,6,7,7a,8,13,13b,13c,14,15-dodecahydro-4H-3,15a-epoxy[1]benzoxepino[6',7':6,7]indeno[1,2-b]indol-4-one](http://img.cochemist.com/ccimg/70600/70553-75-2_b.png)
![2H-1-Benzopyrano[5',6':6,7]indeno[1,2-b]indole-2-methanol,3,4,4a,4b,5,6,6a,7,12,12b,12c,13,14,14a-tetradecahydro-a,a,4a,12b,12c-pentamethyl-,(2S,4aS,4bR,6aS,12bS,12cS,14aS)-](http://img.cochemist.com/ccimg/11100/11024-56-9.png)
![2H-1-Benzopyrano[5',6':6,7]indeno[1,2-b]indole-2-methanol,3,4,4a,4b,5,6,6a,7,12,12b,12c,13,14,14a-tetradecahydro-a,a,4a,12b,12c-pentamethyl-,(2S,4aS,4bR,6aS,12bS,12cS,14aS)-](http://img.cochemist.com/ccimg/11100/11024-56-9_b.png)
![Diphosphoric acid,P-[(2E,6E,10E)-3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraen-1-yl] ester](http://img.cochemist.com/ccimg/6700/6699-20-3.png)
![Diphosphoric acid,P-[(2E,6E,10E)-3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraen-1-yl] ester](http://img.cochemist.com/ccimg/6700/6699-20-3_b.png)
![2H-Pyrano[2'',3'':5',6']benz[1',2':6,7]indeno[1,2-b]indol-3(4bH)-one,5,6,6a,7,12,12b,12c,13,14,14a-decahydro-4b-hydroxy-2-(1-hydroxy-1-methylethyl)-12b,12c-dimethyl-,(2R,4bS,6aS,12bS,12cR,14aS)-](http://img.cochemist.com/ccimg/57200/57186-25-1.png)
![2H-Pyrano[2'',3'':5',6']benz[1',2':6,7]indeno[1,2-b]indol-3(4bH)-one,5,6,6a,7,12,12b,12c,13,14,14a-decahydro-4b-hydroxy-2-(1-hydroxy-1-methylethyl)-12b,12c-dimethyl-,(2R,4bS,6aS,12bS,12cR,14aS)-](http://img.cochemist.com/ccimg/57200/57186-25-1_b.png)