期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 288, 期 47, 页码 34146-34157出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M113.487272
关键词
Bacterial Metabolism; Crystallography; Kinetics; Polyketides; Site-directed Mutagenesis
资金
- Targeted Proteins Research Program of the Ministry of Education, Culture, Sports, Science, and Technology, Japan
- Funding Program for Next Generation World-leading Researchers from the Bureau of Science, Technology, and Innovation Policy, Cabinet Office, Government of Japan
- Grants-in-Aid for Scientific Research [23228003] Funding Source: KAKEN
Background: Type III polyketide synthases (PKSs) show diverse cyclization specificity. Results: A single amino acid substitution in two Azotobacter type III PKSs reversed their cyclization specificity. Crystal structures were determined. Conclusion: The volume of the active site cavity is a crucial determinant of the cyclization specificity. Significance: An important insight into the cyclization specificity of type III PKSs was provided. 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 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. Trp-281 of ArsB corresponded to Gly-284 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 (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 , 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.
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