Structure and Mechanism of the trans-Acting Acyltransferase from the Disorazole Synthase

The 1.51 angstrom resolution X-ray crystal structure of the trans-acyltransferase (AT) from the AT-less disorazole synthase (DSZS) and that of its acetate complex at 1.35 angstrom resolution are reported. Separately, comprehensive alanine-scanning mutagenesis of one of its acyl carrier protein substrates (ACP1 from DSZS) led to the identification of a conserved Asp45 residue on the ACP, which contributes to the substrate specificity of this unusual enzyme. Together, these experimental findings were used to derive a model for the selective association of the DSZS AT and its ACP substrate. With a goal of structurally characterizing the AT-ACP interface, a strategy was developed for covalently cross-linking the active site Ser -> Cys mutant of the DSZS AT to its.ACP substrate and for purifying the resulting AT -> ACP complex to homogeneity. The S86C DSZS AT mutant was found to be functional, albeit with a transacylation efficiency 200-fold lower than that of its wild-type counterpart. Our findings provide new insights as well as new opportunities for high-resolution analysis of an important protein-protein interface in polyketide synthases.