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 polyketide synthases (PKSs), thioesterase domains have been assigned at the C-terminus regions of fungal iterative type I PKSs. We previously overexpressed Aspergillus nidulans wA PKS gene in a heterologous fungal host and identified it to encode a heptaketide naphthopyrone synthase. In addition, expression of C-terminus-modified WA PKS gave heptaketide isocoumarins suggesting that the C-terminus region of WA PKS is involved in the cyclization of the second aromatic ring of naphthopyrone. To unravel the actual function of the C-terminus region, we carried out functional analysis of WA PKS mutants by C-terminus deletion and site-directed mutagenesis.


RESULTS
Only the 32 amino acid deletion from the C-terminus of WA PKS caused product change to heptaketide isocoumarins from heptaketide naphthopyrone, YWA1 1, a product of intact WA PKS. Further C-terminus deletion mutant of WA PKS up to Ser(1967), an active site residue of so far called thioesterase, still produced isocoumarins. Site-directed mutagenesis of amino acid residues in this C-terminus region showed that even a single mutation of S1967A or H2129Q caused production of isocoumarin instead of naphthopyrone. Furthermore, the role of tandem acyl carrier proteins (ACPs), a typical feature of fungal aromatic PKSs, was examined by site-directed mutagenesis and the results indicated that both ACPs can function as ACP independently.


CONCLUSIONS
Claisen-type cyclization is assumed to be involved in formation 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 propose that the so far called TE of these PKSs work not just as TE but as Claisen cyclase.
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
I
I
Fujii
A
A
Watanabe
U
U
Sankawa
Y
Y
Ebizuka
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000201:enzymology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017384:Sequence Deletion
D009097:Multienzyme Complexes