School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
School of Chemistry, Cantock's Close, Clifton, Bristol, BS8 1TS, UK.
Nat Chem Biol. 2013 Nov;9(11):685-692. doi: 10.1038/nchembio.1342. Epub 2013 Sep 22.
Type I polyketide synthases often use programmed β-branching, via enzymes of a 'hydroxymethylglutaryl-CoA synthase (HCS) cassette', to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in acyl carrier proteins (ACPs) where β-branching is known to occur. Substituting ACPs confirmed a correlation of ACP type with β-branching specificity. Although these ACPs often occur in tandem, NMR analysis of tandem β-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modeling and mutagenesis identified ACP helix III as a probable anchor point of the ACP-HCS complex whose position is determined by the core. Mutating the core affects ACP functionality, whereas ACP-HCS interface substitutions modulate system specificity. Our method for predicting β-carbon branching expands the potential for engineering new polyketides and lays a basis for determining specificity rules.
I 型聚酮合酶通常使用“羟甲基戊二酰辅酶 A 合酶（HCS）盒”中的酶进行程序化的β-支化，以在增长的聚酮主链的末端羧酸的第二个碳原子处掺入各种侧链。我们在酰基载体蛋白（ACP）中鉴定出一个已知发生β-支化的强序列基序。取代 ACP 证实了 ACP 类型与β-支化特异性之间的相关性。尽管这些 ACP 通常串联出现，但对串联β-支化 ACP 的 NMR 分析表明没有 ACP-ACP 协同作用，并揭示了保守序列基序形成内部核心而不是暴露的斑块。建模和突变分析确定了 ACP 螺旋 III 作为 ACP-HCS 复合物的一个可能的锚定点，其位置由核心决定。改变核心会影响 ACP 的功能，而 ACP-HCS 界面取代则调节系统特异性。我们预测β-碳原子支化的方法扩展了工程新聚酮的潜力，并为确定特异性规则奠定了基础。
