Re-identification of Aspergillus nidulans wA gene to code for a polyketide synthase of naphthopyrone

Watanabe, Akira; Fujii, Isao; Sankawa, Ushio; Mayorga, Marı́a E.; Timberlake, William E.; Ebizuka, Yutaka

doi:10.1016/s0040-4039(98)80027-0

Cited by 125 publications

(135 citation statements)

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“…The naphthopyrone YWA1 is considered to be a precursor of green spore pigment in aspergilli such as A. nidulans (9). T4HN has been established as the precursor of DHN-melanin in black fungi (5).…”

Section: Discussionmentioning

confidence: 99%

“…A homology search suggested that arp2 and arp1 code for T4HN reductase and scytalone dehydratase, respectively, and that abr1 and abr2 encode multi-copper oxidases. Heterologous expression of alb1 in Aspergillus oryzae demonstrated that the Alb1p polyketide synthase synthesized the heptaketide naphthopyrone YWA1 (8), which was identified as a precursor of the green spore pigment in Aspergillus nidulans (9). The ayg1 gene in the biosynthetic gene cluster, however, showed no significant homology with known genes in the data base.…”

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confidence: 98%

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Hydrolytic Polyketide Shortening by Ayg1p, a Novel Enzyme Involved in Fungal Melanin Biosynthesis

Fujii

Yasuoka

Tsai

et al. 2004

Journal of Biological Chemistry

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The pentaketide 1,3,6,8-tetrahydroxynaphthalene (T4HN) is a key precursor of 1,8-dihydroxynaphthalenemelanin, an important virulence factor in pathogenic fungi, where T4HN is believed to be the direct product of pentaketide synthases. We showed recently the involvement of a novel protein, Ayg1p, in the formation of T4HN from the heptaketide precursor YWA1 in Aspergillus fumigatus. To investigate the mechanism of its enzymatic function, Ayg1p was purified from an Aspergillus oryzae strain that overexpressed the ayg1 gene. The Ayg1p converted the naphthopyrone YWA1 to T4HN with a release of the acetoacetic acid. Although Ayg1p does not show significant homology with known enzymes, a serine protease-type hydrolytic motif is present in its sequence, and serine-specific inhibitors strongly inhibited the activity. To identify its catalytic residues, site-directed Ayg1p mutants were expressed in Escherichia coli, and their enzyme activities were examined. The single substitution mutations S257A, D352A, and H380A resulted in a complete loss of enzyme activity in Ayg1p. These results indicated that the catalytic triad Asp 352 -His 380 -Ser 257 constituted the active-site of Ayg1p. From a Dixon plot analysis, 2-acetyl-1,3,6,8-tetrahydroxynaphthalene was found to be a strong mixed-type inhibitor, suggesting the involvement of an acyl-enzyme intermediate. These studies support the mechanism in which the Ser 257 at the active site functions as a nucleophile to attack the YWA1 side-chain 1 -carbonyl and cleave the carbon-carbon bond between the naphthalene ring and the side chain. Acetoacetic acid is subsequently released from the Ser 257 -O-acetoacetylated Ayg1p by hydrolysis. An enzyme with activity similar to Ayg1p in melanin biosynthesis has not been reported in any other organism.

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Section: Discussionmentioning

confidence: 99%

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confidence: 98%

Hydrolytic Polyketide Shortening by Ayg1p, a Novel Enzyme Involved in Fungal Melanin Biosynthesis

Fujii

Yasuoka

Tsai

et al. 2004

Journal of Biological Chemistry

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“…DHN melanin (Baker, 2008; Chiang et al, 2011;Jørgensen et al, 2011; Langfelder et al, 1998; Tsai et al, 1998Tsai et al, , 2001Watanabe et al, 1999Watanabe et al, , 2000. In addition, all three genomes encode putative multi-copper oxidases that are clustered with the PKS genes and distantly related to Aspergillus fumigatus brown2/Arp2 and F. graminearum gip1 pigment pathway-associated genes (Fig.…”

Section: Predicted Pigment Genesmentioning

confidence: 99%

Phylogenomic analysis of polyketide synthase-encoding genes in Trichoderma

et al. 2012

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Members of the economically important ascomycete genus Trichoderma are ubiquitously distributed around the world. The mycoparasitic lifestyle and plant defence-inducing interactions of Trichoderma spp. make them ideal biocontrol agents. Of the Trichoderma enzymes that produce secondary metabolites, some of which likely play important roles in biocontrol processes, polyketide synthase (PKSs) have garnered less attention than non-ribosomal peptide synthetases such as those that produce peptaibols. We have taken a phylogenomic approach to study the PKS repertoire encoded in the genomes of Trichoderma reesei, Trichoderma atroviride and Trichoderma virens. Our analysis lays a foundation for future research related to PKSs within the genus Trichoderma and in other filamentous fungi. INTRODUCTIONGiven its important roles in bioenergy-related enzyme production and plant and fungal health, the genus Trichoderma has a significant economic footprint (Schuster & Schmoll, 2010). This genus is well known for its opportunistic lifestyle that includes saprotrophy, mycoparasitism, endophytism, and interactions with plants and animals. Because of their ability to antagonize other fungi and stimulate plant defences against phytopathogens, Trichoderma strains have become prominent biocontrol agents (Schuster & Schmoll, 2010). Further highlighting their mycoparasitic lifestyle, sequencing and analysis of the genomes from two mycoparasitic Trichoderma species (Trichoderma virens, Trichoderma atroviride) have shown that these genomes encode a rich catalogue of fungal cell wall-degrading chitinases and a relative paucity of plant cell wall-degrading enzymes (Kubicek et al., 2011;Martinez et al., 2008).As biocontrol agents and pathogens of fungal food crops, secondary metabolite production in Trichoderma spp. is of particular importance. Trichoderma and other filamentous fungi are well known for the production of a diverse array of secondary metabolites. Non-ribosomal peptides and polyketides comprise a major portion of these products, which are synthesized by large proteins composed of various domains for the individual enzymic steps (Cox, 2007). Both broad and genus-specific phylogenomic and functional analysis of polyketide synthase (PKS)-and non-ribosomal peptide synthetase (NRPS)-encoding genes have been performed, which has accelerated the pace at which genes and pathways are being linked to specific secondary metabolites (Bushley & Turgeon, 2010;Bushley et al., 2008;Chiang et al., 2010;Cramer et al., 2006;Gaffoor et al., 2005;Kroken et al., 2003;Kubicek et al., 2011;Lee et al., 2005).Trichoderma spp. are probably best known for their production of peptaibols, which are non-ribosomal peptides with antimicrobial and plant defence-stimulating activity (Viterbo et al., 2007). Relatively less studied in Trichoderma are PKSs. The recent analysis of the three available Trichoderma genome sequences has indicated that the genomes of these organisms encode only a small catalogue of PKSs: T. atroviride and T. virens each encode 18, and Tric...

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“…That is, the bicyclic intermediate 6 partitions either by C-C cyclization in the presence of TE/CLC to 1 or, in its absence, by spontaneous O-C cyclization to give pyrone 4. The Claisen cyclase (CLC) behavior for nonreducing fungal PKS TE domains was first observed in the work of Ebizuka, Fujii, and Watanabe where a normal Claisen product was diverted to its isomeric pyrone when the TE/CLC domain was either truncated (10) or inactivated by site-specific mutagenesis (11). How the PksA TE conducts Claisen cyclization (C-C bond formation) instead of hydrolysis or macrolactonization (O-C bond formation), which is typically observed in FAS and PKS proteins, is not understood.…”

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Structure and function of an iterative polyketide synthase thioesterase domain catalyzing Claisen cyclization in aflatoxin biosynthesis

Korman

Crawford

Labonte

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

103

128

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Polyketide natural products possess diverse architectures and biological functions and share a subset of biosynthetic steps with fatty acid synthesis. The final transformation catalyzed by both polyketide synthases (PKSs) and fatty acid synthases is most often carried out by a thioesterase (TE). The synthetic versatility of TE domains in fungal nonreducing, iterative PKSs (NR-PKSs) has been shown to extend to Claisen cyclase (CLC) chemistry by catalyzing C–C ring closure reactions as opposed to thioester hydrolysis or O–C/N–C macrocyclization observed in previously reported TE structures. Catalysis of C–C bond formation as a product release mechanism dramatically expands the synthetic potential of PKSs, but how this activity was acquired has remained a mystery. We report the biochemical and structural analyses of the TE/CLC domain in polyketide synthase A, the multidomain PKS central to the biosynthesis of aflatoxin B 1 , a potent environmental carcinogen. Mutagenesis experiments confirm the predicted identity of the catalytic triad and its role in catalyzing the final Claisen-type cyclization to the aflatoxin precursor, norsolorinic acid anthrone. The 1.7 Å crystal structure displays an α/β-hydrolase fold in the catalytic closed form with a distinct hydrophobic substrate-binding chamber. We propose that a key rotation of the substrate side chain coupled to a protein conformational change from the open to closed form spatially governs substrate positioning and C–C cyclization. The biochemical studies, the 1.7 Å crystal structure of the TE/CLC domain, and intermediate modeling afford the first mechanistic insights into this widely distributed C–C bond-forming class of TEs.

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Re-identification of Aspergillus nidulans wA gene to code for a polyketide synthase of naphthopyrone

Cited by 125 publications

References 12 publications

Hydrolytic Polyketide Shortening by Ayg1p, a Novel Enzyme Involved in Fungal Melanin Biosynthesis

Hydrolytic Polyketide Shortening by Ayg1p, a Novel Enzyme Involved in Fungal Melanin Biosynthesis

Phylogenomic analysis of polyketide synthase-encoding genes in Trichoderma

Structure and function of an iterative polyketide synthase thioesterase domain catalyzing Claisen cyclization in aflatoxin biosynthesis

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