Structural Basis for Cyclization Specificity of Two Azotobacter Type III Polyketide Synthases

Abstract

Type III polyketide synthases (PKSs) show diverse cyclization specificity. We 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 con- densation 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. Trp-281 of ArsB corresponded to Gly- 284 of ArsC in the amino acid sequence alignment. The ArsB W281Gmutantsynthesizedalkylpyronebutnotalkylresorcinol.In contrast, the ArsC G284W mutant synthesized alkylresorcinol with a smallamountofalkylpyrone. These results indicate that this amino acid residue (Trp-281 of ArsB or Gly-284 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 Å, 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 ofthe 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 ofArsC G284W (and presumably ArsB). This is the first report on the alteration ofcyclization specificity from lacton- ization to aldol condensation for a type III PKS. The ArsCG284W structure is significant as it is the first reportedstructureofamicro- bial resorcinol synthase