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

Fujii, Isao; Yasuoka, Yoshinori; Tsai, Huei‐Fung; Chang, Yun C.; Kwon-Chung, Kyung J.; Ebizuka, Yutaka

doi:10.1074/jbc.m406758200

Cited by 86 publications

(85 citation statements)

References 28 publications

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“…This NRPKS is a member of Group III instead of Group II. This classification is consistent with previous biochemical analysis, which showed that synthesis of THN by WA is a result of chain shortening of the heptaketide product YWA1 (44). The well-studied PksA and other NRPKSs that are involved in the synthesis of C4-C9/C2-C11 cyclized aromatic polyketides are clearly separated into Group IV.…”

Section: Resultssupporting

confidence: 75%

Classification, Prediction, and Verification of the Regioselectivity of Fungal Polyketide Synthase Product Template Domains

Tang

2010

Journal of Biological Chemistry

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The fungal iterative nonreducing polyketide synthases (NRPKSs) synthesize aromatic polyketides, many of which have important biological activities. The product template domains (PT) embedded in the multidomain NRPKSs mediate the regioselective cyclization of the highly reactive polyketide backbones and dictate the final structures of the products. Understanding the sequence-activity relationships of different PT domains is therefore an important step toward the prediction of polyketide structures from NRPKS sequences and can enable the genome mining of hundreds of cryptic NRPKSs uncovered via genome sequencing. In this work, we first performed phylogenetic analysis of PT domains from NRPKSs of known functions and showed that the PT domains can be classified into five groups, with each group corresponding to a unique product size or cyclization regioselectivity. Group V contains the formerly unverified PT domains that were identified as C6-C11 aldol cyclases. The regioselectivity of PTs from this group were verified by product-based assays using the PT domain excised from the asperthecin AptA NRPKS. When combined with dissociated PKS4 minimal PKS, or replaced the endogenous PKS4 C2-C7 PT domain in a hybrid NRPKS, AptA-PT directed the C6-C11 cyclization of the nonaketide backbone to yield a tetracyclic pyranoanthraquinone 4. Extensive NMR analysis verified that the backbone of 4 was indeed cyclized with the expected regioselectivity. The PT phylogenetic analysis was then expanded to include ϳ100 PT sequences from unverified NRPKSs. Using the assays developed for AptA-PT, the regioselectivities of additional PT domains were investigated and matched to those predicted by the phylogenetic classifications.

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Section: Resultssupporting

confidence: 75%

Classification, Prediction, and Verification of the Regioselectivity of Fungal Polyketide Synthase Product Template Domains

Tang

2010

Journal of Biological Chemistry

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“…1), which is then enzymatically converted to T4HN, as shown in Fig. 2C (17,40). YWA1 is metabolized to T4HN by a post-PKS polyketide shortening mechanism (17) involving a protein called Ayglp.…”

mentioning

confidence: 99%

“…This reaction occurs upstream of the T4HN reductase step that is required for the synthesis of scytalone. Other compounds made in A. fumigatus include 1, 3, vermelone, and D2HN (6,17). The reductase that converts T4HN to scytalone can be blocked by a specific pathway inhibitor known as tricyclazole (20).…”

mentioning

confidence: 99%

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New Biosynthetic Step in the Melanin Pathway ofWangiella(Exophiala)dermatitidis: Evidence for 2-Acetyl-1,3,6,8-Tetrahydroxynaphthalene as a Novel Precursor

et al. 2008

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The predominant cell wall melanin of Wangiella dermatitidis, a black fungal pathogen of humans, is synthesized from 1,8-dihydroxynaphthalene (D2HN). An early precursor, 1,3,6,8-tetrahydroxynaphthalene (T4HN), in the pathway leading to D2HN is reportedly produced directly as a pentaketide by an iterative type I polyketide synthase (PKS). In contrast, the bluish-green pigment in Aspergillus fumigatus is produced after the enzyme Ayg1p converts the PKS product, the heptaketide YWA1, to T4HN. Previously, we created a new melanin-deficient mutant of W. dermatitidis, WdBrm1, by random molecular insertion. From this strain, the altered gene WdYG1 was cloned by a marker rescue strategy and found to encode WdYg1p, an ortholog of Ayg1p. In the present study, two gene replacement mutants devoid of the complete WdYG1 gene were derived to eliminate the possibility that the phenotype of WdBrm1 was due to other mutations. Characterization of the new mutants showed that they were phenotypically identical to WdBrm1. Chemical analyses of mutant cultures demonstrated that melanin biosynthesis was blocked, resulting in the accumulation of 2-acetyl-1,3,6,8-tetrahydroxynaphthalene (AT4HN) and its oxidative product 3-acetylflaviolin in the culture media. When given to an albino W. dermatitidis strain with an inactivated WdPKS1 gene, AT4HN was mostly oxidized to 3-acetylflaviolin and deacetylated to flaviolin. Under reduced oxygen conditions, cell-free homogenates of the albino converted AT4HN to D2HN. This is the first report of evidence that the hexaketide AT4HN is a melanin precursor for T4HN in W. dermatitidis.Melanins are dark pigments widely produced by fungi and other organisms. In fungi, they are frequently found in the cell wall. While not essential for growth and development, these complex polymers seem to enhance the survival and competitive abilities of fungi in certain environments. They are composed of various types of phenolic monomers and are often complexed with protein and, less often, carbohydrates (12,22,29). The melanins in fungi are named according to their composition and the way they are synthesized and include dihydroxyphenylalanine melanin, catechol melanin, ␥-glutaminyl-4-hydroxybenzene melanin, and 1,8-dihydroxynaphthalene (D2HN) melanin (23, 26). The best characterized of these fungal melanins is probably D2HN melanin, which is synthesized by related polyketide pathways ( Fig. 1 and 2A and B). The D2HN melanin pathways start with one acetyl-coenzyme A (acetyl-CoA) molecule and four malonyl-CoA molecules, or solely with malonyl-CoA molecules, which undergo a head-totail joining and cyclization catalyzed by an iterative type I polyketide synthase (PKS) to initially form 1,3,6,8-tetrahydroxynaphthalene (T4HN) (16). From T4HN, multiple sequential enzyme-catalyzed steps produce D2HN, which is then polymerized to form melanin by a poorly characterized oxidase/laccase reaction (5,7,22).In Colletotrichum lagenarium, T4HN is made directly from malonyl-CoA by PKS1p, as shown in Fig. 2B (16). In contrast, in the bluis...

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“…21 The translational product PKS4 is capable of producing aromatic polyketides using acylCoAs as substrates as reported by Tang et al 21 This PKS4 naturally found in G. fujikuroi is responsible for the biosynthesis of bikaverin (1, Figure 6), a fungal polyketide exhibiting anticancer activity. Mirroring the YWA1 biosynthetic pathway, 25 a proposed mechanism for assembling the aromatic rings involves an aldol condensation reaction between C2 and C7 of the first ring followed by a Claisen condensation between C10 and C1 to form the pentaketide 1, 3, 6, 8-tetrahydroxynaphthalene 26,27 scaffold by the thioesterase/Claisenlike cyclase (TE/CLC) domain in the enzyme which is finished off by a pyrone formation reaction. Subsequently, compound 2 is provided through a dehydration followed by cyclization between C12 and C17.…”

Section: Aromatic Polyketides Biosynthesized Using Pks4mentioning

confidence: 99%

A comprehensive overview on genomically directed assembly of aromatic polyketides and macrolide lactones using fungal megasynthases

et al. 2010

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Fungal polyketide synthases (PKSs) catalyze a carbon-carbon bond forming reaction in an iterative manner using a variety of acyl-CoA molecules as substrates when biosynthesizing complex polyketides. Although most members from this class of natural products exhibit notable biological activities, often they are naturally produced in trace levels or cultivation of the analyteproducing organism is less than feasible. Appropriately, to contend with the former challenge, one must identify any translational bottleneck and perform functional analysis of the associated enzymes. In recent years, many gene clusters purportedly responsible for biosynthesizing polyketides have been identified and cataloged from a variety of fungal genomes including genes coding for iterative PKSs, particulary bikaverin, zearalenone and hypothemycin biosynthetic enzymes. Mounting appreciation of these highly specific codons and their translational consequence will afford scientists the ability to anticipate the fungal metabolite by correlating an organism's genomic cluster to an appropriate biosynthetic system. It was observed in recent reports, the successful production of these recombinant enzymes using an Escherichia coli expression system which in turn conferred the anticipated metabolite in vitro. This review will focus on iterative PKSs responsible for biosynthesizing bikaverin, zearalenone and hypothemycin, and expand on befitting enzymatic reaction mechanisms and development of a highly versatile system that could potentially generate biologically active compounds.

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

Cited by 86 publications

References 28 publications

Classification, Prediction, and Verification of the Regioselectivity of Fungal Polyketide Synthase Product Template Domains

Classification, Prediction, and Verification of the Regioselectivity of Fungal Polyketide Synthase Product Template Domains

New Biosynthetic Step in the Melanin Pathway ofWangiella(Exophiala)dermatitidis: Evidence for 2-Acetyl-1,3,6,8-Tetrahydroxynaphthalene as a Novel Precursor

A comprehensive overview on genomically directed assembly of aromatic polyketides and macrolide lactones using fungal megasynthases

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