Identification of Claisen cyclase domain in fungal polyketide synthase WA,a naphthopyrone synthase of Aspergillus nidulans

Background: Based on the homology with fatty acid synthases and bacterial p olyketide synthases(PKSs), thioesterase domains have been assigned at the C -terminus regions of fungal iterative type I PKSs, We previously overexpres sed Aspergillus nidulans wA PKS gene in a heterologous fungal host and iden tified it to encode a heptaketide naphthopyrone synthase. In addition, expr ession of C-terminus-modified WA PKS gave heptaketide isocoumarins suggesti ng that the C-terminus region of WA PKS is involved in the cyclization of t he second aromatic ring of naphthopyrone. To unravel the actual function of the C-terminus region, we carried out functional analysis of WA PKS mutant s by C-terminus deletion and site-directed mutagenesis. Results: Only the 32 amino acid deletion from the C-terminus of WA PKS caus ed product change to heptaketide isocoumarins from heptaketide naphthopyron e, YWA1 1, a product of intact WA PKS. Further C-terminus deletion mutant o f WA PKS up to Ser(1967), an active site residue of so far called thioester ase, still produced isocoumarins. Site-directed mutagenesis of amino acid r esidues in this C-terminus region showed that even a single mutation of S19 67A or H2129Q caused production of isocoumarin instead of naphthopyrone. Fu rthermore, the role of tandem acyl carrier proteins (ACPs), a typical featu re of fungal aromatic PKSs, was examined by site-directed mutagenesis and t he results indicated that both ACPs can function as ACP independently. Conclusions: Claisen-type cyclization is assumed to be involved in formatio n of aromatic compounds by some fungal type I PKSs. These PKSs have a quite identical architecture of active site domain organization; beta -ketoacyl synthase, acyltransferase, tandem ACPs and thioesterase (TE) domains. Since the C-terminus region of WA PKS of this type was determined to be involved in Claisen-type cyclization of the second ring of naphthopyrone, we propos e that the so far called TE of these PKSs work not just as TE but as Claise n cyclase. (C) 2001 Elsevier Science Ltd. All rights reserved.