Structure and Mechanism of the trans-Acting Acyltransferase from the Disorazole Synthase
- 27 June 2011
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 50 (30), 6539-6548
- https://doi.org/10.1021/bi200632j
Abstract
The 1.51 Å 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 Å 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.This publication has 36 references indexed in Scilit:
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