Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Department of Biotechnology, The Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Acta Crystallogr D Struct Biol. 2022 Sep 1;78(Pt 9):1171-1179. doi: 10.1107/S2059798322007434. Epub 2022 Aug 30.
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.
酮基合酶（KSs）在脂肪酸生物合成中使用两步乒乓反应机制催化重要的碳-碳键形成反应。在大肠杆菌中，有两种同源二聚体延伸 KSs，FabB 和 FabF，它们具有重叠的底物选择性。然而，FabB 对于不饱和脂肪酸（UFAs）的生物合成是必需的，这些不饱和脂肪酸是细胞在没有外源 UFAs 的情况下生存所必需的。此外，FabB 对长于 12 个碳原子的底物的活性降低，而 FabF 则有效地催化饱和 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，解决了两个与 ACP 复合的 FabB 的交联晶体结构，该 ACP 用近似催化步骤的长链脂肪酸交联探针进行了功能化。这两种同源二聚体结构都具有不对称的底物结合口袋，表明当与 C14 和 C16 酰基链结合时，两个 FabB 单体之间存在协同关系。此外，这些结构捕获了活性位点门控残基 Phe392 的一种异常旋转体，这可能代表了在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捉和阐明伴随 KS 介导的催化的构象转变的实用性。
