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.
Fujii
I
I
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Watanabe
A
A
Sankawa
U
U
Ebizuka
Y
Y
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