Polyketides are important natural products that include numerous toxins, antibiotics, a variety of therapeutic compounds, fungal melanins, and other pigments. Polyketides have attracted great attention because of their biosynthetic complexity and importance in the pharmaceutical industry. Extensive molecular genetic studies of polyketide biosynthesis have been carried out in actinomycetes and Gram-positive bacteria. Microbial polyketides are generally assembled by three types of polyketide synthase (PKS) 1 (1). Type I modular PKSs are large multifunctional polypeptides that consist of a number of modular units (modules), each of which is responsible for single -ketoacyl condensation and the following reduction steps. Because modules are used sequentially and nonrepetitively, the number of modules determines the length of the carbon backbones of reduced complex-type polyketides. On the other hand, type II PKSs consist of several single-function enzymes that are used iteratively for bacterial aromatic polyketides, and the determinants for polyketide skeletons are still unclear. Type III are small plant chalcone synthase-type PKSs, and RppA was identified as the first microbial PKS of this class (2). Fungal PKSs fall into type I, consisting of a single large polypeptide with a set of active site domains similar to the modular type I PKSs, but they work iteratively to produce their specific products including both aromatic and reduced complextype compounds. Thus they might be classified as an independent group of PKSs (3). Although several PKS genes have been identified from various fungal species (4 -15), exactly how a fungal PKS synthesizes a specific polyketide remains unclear. In general, PKS is the sole determinant of the chain length and cyclization pattern of a polyketide. However, a recent report showed that an accessory protein (LovC) is needed to enable the PKS (LovB) of Aspergillus terreus to synthesize a fulllength polyketide precursor, dihydromonacolin L, for lovastatin biosynthesis. This suggests that fungal PKS might be of a more complex nature than the modular PKS. (9).Pentaketide melanins have been shown to be important virulence factors in fungal species pathogenic to plants or humans (13, 16 -18). It is generally believed that acetyl-CoA and malonyl-CoA are the starter and extender of polyketide synthases involved in the fungal 1,8-dihydroxynaphthalene (DHN)-melanin pathway. However, recently malonyl-CoA was demonstrated as the sole starter of Colletotrichum lagenarium PKS1 for production of the precursor 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN) (see Fig. 1A) (19). The pentaketide, 1,3,6,8-THN, is then reduced by 1,3,6,8-THN reductase to scytalone, which is subsequently converted to DHN following the dehydration and reduction steps. Finally, DHN is polymerized to form DHN-melanin. Tricyclazole, a fungicide, specifically inhibits both THN reductase reactions involved in the DHNmelanin pathway (see Fig. 1A) (20). To date, the genes encoding
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.
Polyketides are one of the largest and most important groups of natural products. In spite of their structural diversity, the initial reactions of polyketide biosynthesis follow a common scheme that condensation of acyl primer with malonate extension units to form-polyketomethylene intermediates and their cyclizations catalyzed by so called polyketide synthases (PKSs) 1). Although molecular genetic analysis of bacterial polyketide biosynthesis genes have been extensively carried out, only several PKS genes have been cloned from eukaryotic filamentous fungi, which are another rich source of polyketides, especially, aromatic compounds 2). All fungal PKS genes so far cloned code for iterative multifunctional type I PKS polypeptides. So far, little has been known how fungal PKSs control their reactions, that is, how to regulate the chain-lengths of-polyketomethylene intermediates and their cyclizations. Thus, expression and functional analysis of fungal iterative type I PKSs were carried out. Aspergillus terreus ATX 3) 6-Methylsalicylic acid synthase (MSAS) of Penicillium patulum was the first fungal PKS of which enzyme activity was detected and the gene was cloned 4,5). Hopwood et al. indicated the presence of
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