STRUCTURAL SIMILARITIES BETWEEN 6-METHYLSALICYLIC ACID SYNTHASE FROM PENICILLIUM-PATULUM AND VERTEBRATE TYPE-I FATTY-ACID SYNTHASE - EVIDENCE FROM THIOL MODIFICATION STUDIES

6-Methylsalicylic acid synthase, the multifunctional enzyme complex th at catalyzes the biosynthesis of the tetraketide 6-methylsalicylic aci d,was modified by thiol-specific inhibitors and crosslinking reagents. Treatment with 1,3-dibromopropan-2-one caused rapid enzyme inactivati on and formation of cross-linked dimers, Analysis by SDS-PAGE, density gradient ultracentrifugation, and secondary modification with [C-14]i odoacetamide showed that two types of cross-linked dimers were formed. Peptides derived from native and 1,3-dibromopropan[2-C-14]one-treated enzyme were isolated by SDS-PAGE and N-terminally sequenced. The sequ ences of the two N-termini from cross-linked peptides were located in the nucleotide-derived amino acid sequence and found to arise from the beta-ketoacyl synthase and acyl carrier protein components of the 6-m ethylsalicylic acid synthase subunit. Acetyl-CoA protected the enzyme from both inactivation and cross-linking by binding to the reactive cy steine of the beta-ketoacyl synthase component. Malonyl-CoA protected against cross-linking by binding to the thiol moiety of the 4'-phospho pantetheine prosthetic group of the acyl carrier protein. Formation of a mixed disulfide on treatment with 5,5'-dithiobis (2-nitrobenzoic ac id) indicated that these two types of thiol residue are positioned clo se to each other in the active enzyme. From these studies, it was conc luded that two pairs of functional dimers are present in the 6-methyls alicylic acid synthase tetramer and that, within each dimer, the beta- ketoacyl synthase and acyl carrier protein components are juxtaposed t o allow the respective cysteine (residue 204) and 4'-phosphopantethein e thiols to interact during condensation, This spatial arrangement of thiols at the condensing active site is analogous to that found in typ e I vertebrate fatty acid synthases and other polyketide synthases.