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Structural and mechanistic determinants of biosynthetic functional diversity in the type III polyketide synthase superfamily

Posted on:2006-06-21Degree:Ph.DType:Dissertation
University:University of California, San DiegoCandidate:Austin, Michael BFull Text:PDF
GTID:1450390008973986Subject:Chemistry
Abstract/Summary:
The architecturally simple type III polyketide synthases (PKSs) are homodimeric ketosynthase domains that utilize a conserved Cys-His-Asn catalytic triad within an internal active site cavity. These multi-functional enzymes iteratively condense acetyl units derived from malonyl-CoA to a CoA-linked substrate, and typically terminate polyketide chain extension via intramolecular cyclization. Type III PKSs vary in their substrate selection, number of polyketide extensions catalyzed, and mechanism of intramolecular polyketide cyclization. Previous work suggested that type III PKS substrate and product diversity stems from steric modulation of chemically inert residues lining the active site cavity. Here, a thorough homology-based bioinformatic evaluation of each functionally distinct type III PKS produced detailed predictions of residues responsible for most type III PKS catalytic specificities, while also focusing our mechanistic attention upon two enzyme families most likely to utilize design principles other than 'steric modulation'. In each of these latter cases, elucidation of these enzymes' crystal structures revealed unanticipated conformational rearrangements of the typical type III PKS fold, and also gave rise to novel mechanistic hypotheses for subsequent and future experimentation. First, protein engineering guided by the first stilbene synthase (STS) crystal structure identified the programmable "aldol switch" emergent hydrogen bond network responsible for this enzyme's alternative cyclization specificity. I then solved the crystal structure of Streptomyces coelicolor 1,3,6,8-tetrahydroxynaphthalene synthase (THNS), which synthesizes two fused hydroxylated rings from five molecules of malonyl-CoA. This first bacterial type III PKS crystal structure revealed an unanticipated additional active site tunnel that provided the first explanation for these enzymes' cryptic activities when given long acyl starters. Subsequent mutagenesis of THNS generated additional novel hypotheses regarding enzymatic control of reactive polyketide intermediates. Finally, my recent bioinformatic discovery of type III PKS enzymes covalently incorporated into huge FAS-like fusion proteins promises to greatly increase the scope of biosynthetic diversity within the near reach of metabolic engineering. Notably, preliminary structural and functional analysis of these unprecedented Dictyostelium discoideum type III PKS domains has confirmed their homodimeric assembly and iterative catalytic activity.
Keywords/Search Tags:Type III, III PKS, Synthase, Catalytic, Mechanistic, Diversity, Active site cavity
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