3vs9
From Proteopedia
Crystal structure of type III PKS ArsC mutant
Structural highlights
FunctionPublication Abstract from PubMedType III polyketide synthases (PKSs) show diverse cyclization specificity. We have previously characterized two Azotobacter type III PKSs (ArsB and ArsC) with different cyclization specificity. ArsB and ArsC, which share a high sequence identity (71%), produce alkylresorcinols and alkylpyrones, through aldol condensation and lactonization of the same polyketomethylene intermediate, respectively. Here we identified a key amino acid residue for the cyclization specificity of each enzyme by site-directed mutagenesis. Trp281 of ArsB corresponded to Gly284 of ArsC in the amino acid sequence alignment. The ArsB W281G mutant synthesized alkylpyrone, but not alkylresorcinol. In contrast, the ArsC G284W mutant synthesized alkylresorcinol with a small amount of alkylpyrone. These results indicate that this amino acid residue (Trp281 of ArsB or Gly284 of ArsC) should occupy a critical position for the cyclization specificity of each enzyme. We then determined crystal structures of the wild-type and G284W ArsC proteins at resolutions of 1.76 and 1.99 A, respectively. Comparison of these two ArsC structures indicates that the G284W substitution brings a steric wall to the active site cavity, resulting in a significant reduction of the cavity volume. We postulate that the polyketomethylene intermediate can be folded to a suitable form for aldol condensation only in such a relatively narrow cavity of ArsC G284W (and presumably ArsB). This is the first report on the alteration of cyclization specificity from lactonization to aldol condensation for a type III PKS. The ArsC G284W structure is significant as it is the first reported structure of a microbial resorcinol synthase. Structural basis for cyclization specificity of two Azotobacter type III polyketide synthases: a single amino acid substitution reverses their cyclization specificity.,Satou R, Miyanaga A, Ozawa H, Funa N, Katsuyama Y, Miyazono KI, Tanokura M, Ohnishi Y, Horinouchi S J Biol Chem. 2013 Oct 7. PMID:24100027[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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