Department of Chemical Engineering, Department of Chemistry, and §Department of Biochemistry, Stanford University , Stanford, California 94305, United States.
Biochemistry. 2011 Aug 2;50(30):6539-48. doi: 10.1021/bi200632j. Epub 2011 Jul 5.
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.
报道了“无酰基转移酶”（AT）的反酰基转移酶（AT）的 1.51 Å 分辨率 X 射线晶体结构，以及其乙酸复合物的 1.35 Å 分辨率结构。此外，对其酰基载体蛋白底物之一（来自 DSZS 的 ACP1）进行全面的丙氨酸扫描诱变，确定了 ACP 上一个保守的 Asp45 残基，该残基有助于该不寻常酶的底物特异性。这些实验结果共同为 DSZS AT 与其 ACP 底物的选择性结合提供了模型。为了对 AT-ACP 界面进行结构表征，开发了一种将 DSZS AT 的活性位点 Ser → Cys 突变体与 ACP 底物共价交联的策略，并将得到的 AT-ACP 复合物纯化为均相。发现 S86C DSZS AT 突变体是功能性的，尽管其反酰基转移效率比其野生型低 200 倍。我们的发现为聚酮合酶中重要的蛋白质-蛋白质界面的高分辨率分析提供了新的见解和新的机会。
