PDF(Pubmed)
Abstract：
Ketosynthases (KSs) catalyse essential carbon-carbon bond-forming reactions in fatty-acid biosynthesis using a two-step, ping-pong reaction mechanism. In Escherichia coli, there are two homodimeric elongating KSs, FabB and FabF, which possess overlapping substrate selectivity. However, FabB is essential for the biosynthesis of the unsaturated fatty acids (UFAs) required for cell survival in the absence of exogenous UFAs. Additionally, FabB has reduced activity towards substrates longer than 12 C atoms, whereas FabF efficiently catalyses the elongation of saturated C14 and unsaturated C16:1 acyl-acyl carrier protein (ACP) complexes. In this study, two cross-linked crystal structures of FabB in complex with ACPs functionalized with long-chain fatty-acid cross-linking probes that approximate catalytic steps were solved. Both homodimeric structures possess asymmetric substrate-binding pockets suggestive of cooperative relationships between the two FabB monomers when engaged with C14 and C16 acyl chains. In addition, these structures capture an unusual rotamer of the active-site gating residue, Phe392, which is potentially representative of the catalytic state prior to substrate release. These structures demonstrate the utility of mechanism-based cross-linking methods to capture and elucidate conformational transitions accompanying KS-mediated catalysis at near-atomic resolution.
摘要：
酮合成酶（KSs）使用两步法催化脂肪酸生物合成中必需的碳-碳键形成反应，乒乓反应机理。在大肠杆菌中，有两个同源二聚体延伸的KSs，FabB和FabF,具有重叠的底物选择性。然而,FabB对于在不存在外源UFA的情况下细胞存活所需的不饱和脂肪酸(UFA)的生物合成是必需的。此外,FabB对超过12个C原子的底物的活性降低，而FabF有效催化饱和C14和不饱和C16：1酰基-酰基载体蛋白（ACP）复合物的延伸。在这项研究中,FabB的两个交联晶体结构与用近似催化步骤的长链脂肪酸交联探针官能化的ACP复合。两种同二聚体结构都具有不对称的底物结合袋，这表明当与C14和C16酰基链接合时，两个FabB单体之间存在合作关系。此外,这些结构捕获了活性位点门控残基的不寻常旋转异构体，Phe392,其潜在地代表底物释放之前的催化状态。这些结构证明了基于机制的交联方法在近原子分辨率下捕获和阐明伴随KS介导的催化的构象转变的实用性。
