Abstract:
:6-Methylsalicylic acid synthase, the multifunctional enzyme complex that catalyzes the biosynthesis of the tetraketide 6-methylsalicylic acid, was modified by thiol-specific inhibitors and cross-linking reagents. Treatment with 1,3-dibromopropan-2-one caused rapid enzyme inactivation and formation of cross-linked dimers. Analysis by SDS-PAGE, density gradient ultracentrifugation, and secondary modification with [14C]iodoacetamide showed that two types of cross-linked dimers were formed. Peptides derived from native and 1,3-dibromopropan[2-14C]one-treated enzyme were isolated by SDS-PAGE and N-terminally sequenced. The sequences 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-methylsalicylic acid synthase subunit. Acetyl-CoA protected the enzyme from both inactivation and cross-linking by binding to the reactive cysteine of the beta-ketoacyl synthase component. Malonyl-CoA protected against cross-linking by binding to the thiol moiety of the 4'-phosphopantetheine prosthetic group of the acyl carrier protein. Formation of a mixed disulfide on treatment with 5,5'-dithiobis(2-nitrobenzoic acid) indicated that these two types of thiol residue are positioned close to each other in the active enzyme. From these studies, it was concluded that two pairs of functional dimers are present in the 6-methylsalicylic acid synthase tetramer and that, within each dimer, the beta-ketoacyl synthase and acyl carrier protein components are juxtaposed to allow the respective cysteine (residue 204) and 4'-phosphopantetheine thiols to interact during condensation. This spatial arrangement of thiols at the condensing active site is analogous to that found in type I vertebrate fatty acid synthases and other polyketide synthases.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Child CJ,Spencer JB,Bhogal P,Shoolingin-Jordan PMdoi
10.1021/bi960422esubject
Has Abstractpub_date
1996-09-24 00:00:00pages
12267-74issue
38eissn
0006-2960issn
1520-4995pii
bi960422ejournal_volume
35pub_type
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