Search

Research is presented describing the stereospecific hydration of alpha,beta-unsaturated acyl-CoA thiolesters catalyzed by enoyl-CoA hydratase. The absolute stereospecificity for the enzyme-catalyzed reaction is calculated.

The chemical synthesis of paeoniflorin, the alpha-glucoside of paeoniflorigenin, and of plus- or minus-paeoniflorigenin has been accomplished. Paeoniflorin is a unique complex terpenoid from the roots of Paeonia lactiflora, the Chinese paeony, commonly employed in conventional Chinese medicine. It has been associated with intriguing neuromuscular, antiinflammatory, sedative and anticoagulant activities.

A dogma for DNA polymerase catalysis is that the enzyme binds DNA first, followed by MgdNTP. This mechanism contributes to the selection of correct dNTP by Watson–Crick base pairing, but it cannot explain how low-fidelity DNA polymerases overcome Watson–Crick base pairing to catalyze non-Watson–Crick dNTP incorporation. DNA polymerase X from the deadly African swine fever virus (Pol X) is a half-sized repair polymerase that catalyzes efficient dG:dGTP incorporation in addition to correct repair. Here we report the use of solution structures of Pol X in the free, binary (Pol X:MgdGTP), and ternary (Pol X:DNA:MgdGTP with dG:dGTP non-Watson–Crick pairing) forms, along with functional analyses, to show that Pol X uses multiple unprecedented strategies to achieve the mutagenic dG:dGTP incorporation. Unlike high fidelity polymerases, Pol X can prebind purine MgdNTP tightly and undergo a specific conformational change in the absence of DNA. The prebound MgdGTP assumes an unusual syn conformation stabilized by partial ring stacking with His115. Upon binding of a gapped DNA, also with a unique mechanism involving primarily helix aE, the prebound syn-dGTP forms a Hoogsteen base pair with the template anti-dG. Interestingly, while Pol X prebinds MgdCTP weakly, the correct dG:dCTP ternary complex is readily formed in the presence of DNA. H115A mutation disrupted MgdGTP binding and dG:dGTP ternary complex formation but not dG:dCTP ternary complex formation. The results demonstrate the first solution structural view of DNA polymerase catalysis, a unique DNA binding mode, and a novel mechanism for non-Watson–Crick incorporation by a low-fidelity DNA polymerase. [ABSTRACT FROM AUTHOR]