Showing papers by "Yutaka Ebizuka published in 2002"
TL;DR: RppA was also found to synthesize a C-methylated pyrone, 3,6-dimethyl-4-hydroxy-2-pyr one, by using acetoacetyl-CoA as the starter and methylmalonyl- coA as an extender, demonstrating the broad substrate specificity of RppA.
90 citations
TL;DR: Theexpression of aknX in Escherichia coli indicates that AknX and related gene products such as DnrG and SnoaB are involved in the formation of aklanonic acid from its anthrone precursor, as suggested by their homology with TcmH and ActVA6.
Abstract: In streptomycete anthracycline biosynthetic gene clusters, small open reading frames are located just upstream of minimal polyketide synthase genes. aknX is such a gene found in the aklavinone-aclacinomycin biosynthetic gene cluster of Streptomyces galilaeus . In order to identify its function, the aknX gene was expressed in Escherichia coli . The cell extract prepared from E. coli cells overexpressing AknX protein exhibited anthrone oxygenase activity, which converted emodinanthrone to anthraquinone emodin. This indicates that AknX and related gene products such as DnrG and SnoaB are involved in the formation of aklanonic acid from its anthrone precursor, as suggested by their homology with TcmH and ActVA6. The AknX protein fused with a His 6 tag was efficiently purified to homogeneity by Ni 2+ affinity and anion-exchange column chromatography. The native molecular mass of AknX was estimated to be 42 kDa by gel filtration. Thus, native AknX is considered to have a homotrimeric subunit structure. AknX, like TcmH and ActVA6, possesses no apparent prosthetic group for oxygen activation. Site-directed mutagenesis was carried out to identify the key amino acid residue(s) involved in the oxygenation reaction. Of seven AknX mutants expressed, the W67F mutant showed significantly reduced oxygenase activity, suggesting the important role of the W67 residue in the AknX reaction. A possible mechanism for the reaction via peroxy anion intermediate is proposed.
62 citations
TL;DR: The present result is the first demonstration of the functional expression of OSCs from monocotyledonous plants, and two cDNAs were proved to encode a CAS and a multifunctional triterpene synthase, respectively.
Abstract: Costus speciosus produces a large quantity of steroidal glycosides derived from the sole aglycone, diosgenin. Cycloartenol, a product of oxidosqualene cyclase (OSC), is postulated to be a common intermediate for phytosterols of primary metabolism and diosgenin of secondary metabolism, possibly providing a metabolic branch point. Two cDNAs, CSOSC1 and CSOSC2, were cloned from C. speciosus by RT-PCR and cDNA library screening. Both cDNAs encode 759 amino acids with high mutual identity (74%), resembling (>55% identity) the known OSCs. Phylogenetic tree analysis indicated that the gene products occupy distinct positions from those of cycloartenol synthases (CASs) and triterpene synthases from dicotyledonous plants. By functional expression in yeast, CSOSC1 and CSOSC2 were proved to encode a CAS and a multifunctional triterpene synthase, respectively. The present result is the first demonstration of the functional expression of OSCs from monocotyledonous plants.
48 citations
TL;DR: In this article, site-directed mutagenesis of the amino acid residues that are likely to form the active-site cavity revealed that the aromatic ring of Tyr(224) is essential for RppA to select malonyl-CoA as a starter substrate.
Abstract: RppA, which belongs to the type III polyketide synthase family, catalyses the synthesis of 1,3,6,8-tetrahydroxynaphthalene (THN), which is the key intermediate of melanin biosynthesis in the bacterium Streptomyces griseus. The reaction of THN synthesis catalysed by RppA is unique in the type III polyketide synthase family, in that it selects malonyl-CoA as a starter substrate. The Cys-His-Asn catalytic triad is also present in RppA, as in plant chalcone synthases, as revealed by analyses of active-site mutants having amino acid replacements at Cys(138), His(270) and Asn(303) of RppA. Site-directed mutagenesis of the amino acid residues that are likely to form the active-site cavity revealed that the aromatic ring of Tyr(224) is essential for RppA to select malonyl-CoA as a starter substrate, since substitution of Tyr(224) by amino acids other than Phe and Trp abolished the ability of RppA to accept malonyl-CoA as a starter, whereas the mutant enzymes Y224F and Y224W were capable of synthesizing THN via the malonyl-CoA-primed reaction. Of the site-directed mutants generated, A305I was found to produce only a triketide pyrone from hexanoyl-CoA as starter substrate, although wild-type RppA synthesizes tetraketide and triketide pyrones in the hexanoyl-CoA-primed reaction. The kinetic parameters of Ala(305) mutants and identification of their products showed that the substitution of Ala(305) by bulky amino acid residues restricted the number of elongations of the growing polyketide chain. Both Tyr(224) (important for starter substrate selection) and Ala(305) (important for intermediate elongation) were found to be conserved in three other RppAs from Streptomyces antibioticus and Streptomyces lividans.
46 citations
TL;DR: Findings indicate broad substrate specificity of the WA C-terminal domain for Claisen-type cyclization and an unprecedented chain length determination mechanism of fungal aromatic PKSs.
37 citations
TL;DR: To investigate the mechanism of stilbene synthase (STS) reaction, the origin of the aromatic protons of resveratrol B-ring was examined using STS from Arachis hypogaea expressed in Escherichia coli and deuterated malonyl CoA to detect decarboxylation earlier than aromatization.
5 citations
TL;DR: In this paper, cDNA cloning of oxidosqualene cyclases and analysis of their product specificity were carried out, and over twenty-five OSC clones were obtained, and their enzyme function established by expression in yeast.
Abstract: Cyclization of oxidosqualene into tetra- and pentacyclic carbon skeleton of sterols and triterpenes, catalyzed by oxidosqualene cyclases (OSCs), is one of the most complex and fascinating reactions found in nature. OSCs generate multiple stereogenic centers in a single reaction, and are responsible for the diverse triterpene skeletons. In order to investigate the origin of structural diversity of triterpene skeletons, cDNA cloning of OSCs and analysis of their product specificity were carried out. From triterpene producing plants, over twenty-five OSC clones were obtained, and their enzyme function established by expression in yeast. They included cycloartenol, cucurbitadienol, lupeol, β-amyrin, isomultiflorenol and mixed amyrin synthases. Studies of chimeric proteins between β-amyrin synthase and lupeol synthase, and mutant proeins constructed by site directed mutagenesis identified the amino acid residues responsible for their product specificity. Trp 259 of β-amyrin synthase (PNY) was identified to be the critical residue controlling β-amyrin formation. In further mutation studies, PNY Y 261 H mutant produced dammara-18, 21-dien-3β-ol (as a 3 : 5 mixture of E/Z isomer at Δ18) together with minor amount of dammara-18 (28), 21-dien-3β-ol. These triterpenes have not been reported from nature, and therefore, could be categorized as “unnatural” natural products. The results of this study opened up the possibility of generating new triterpene synthases with additional novel functions through point mutations.
1 citations
TL;DR: Chemical identification of products confirmed their functions and gave some insights into how their reactions are controlled.
Abstract: All fungal polyketide synthase (PKS) genes so far cloned code for iterative type I PKSs that are different from bacterial modular type I PKSs and type II PKSs. In order to clarify how fungal PKSs control their reactions to produce specific product compounds, fungal PKSs such as Aspergillus terreus ATX, Aspergillus nidulans WA, Colletotrichum lagenarium PKS1, Aspergillus fumagatus Alb1p and their derivatives, were expressed under α-amylase promoter in heterologous host Aspergillus oryzae. Chemical identification of products confirmed their functions and gave some insights into how their reactions are controlled.
1 citations
TL;DR: In this article, cDNA cloning of oxidosqualene cyclases and analysis of their product specificity were carried out, and over twenty-five OSC clones were obtained, and their enzyme function established by expression in yeast.
Abstract: Cyclization of oxidosqualene into tetra- and pentacyclic carbon skeleton of sterols and triterpenes, catalyzed by oxidosqualene cyclases (OSCs), is one of the most complex and fascinating reactions found in nature. OSCs generate multiple stereogenic centers in a single reaction, and are responsible for the diverse triterpene skeletons. In order to investigate the origin of structural diversity of triterpene skeletons, cDNA cloning of OSCs and analysis of their product specificity were carried out. From triterpene producing plants, over twenty-five OSC clones were obtained, and their enzyme function established by expression in yeast. They included cycloartenol, cucurbitadienol, lupeol, β-amyrin, isomultiflorenol and mixed amyrin synthases. Studies of chimeric proteins between β-amyrin synthase and lupeol synthase, and mutant proeins constructed by site directed mutagenesis identified the amino acid residues responsible for their product specificity. Trp 259 of β-amyrin synthase (PNY) was identified to be the critical residue controlling β-amyrin formation. In further mutation studies, PNY Y 261 H mutant produced dammara-18, 21-dien-3β-ol (as a 3 : 5 mixture of E/Z isomer at Δ18) together with minor amount of dammara-18 (28), 21-dien-3β-ol. These triterpenes have not been reported from nature, and therefore, could be categorized as “unnatural” natural products. The results of this study opened up the possibility of generating new triterpene synthases with additional novel functions through point mutations.