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Mechanism-based cross-linking probes capture the Escherichia coli ketosynthase FabB in conformationally distinct catalytic states
Acta Crystallographica Section D ( IF 2.6 ) Pub Date : 2022-08-30 , DOI: 10.1107/s2059798322007434
Aochiu Chen 1 , Jeffrey T Mindrebo 1 , Tony D Davis 1 , Woojoo E Kim 1 , Yohei Katsuyama 2 , Ziran Jiang 1 , Yasuo Ohnishi 2 , Joseph P Noel 3 , Michael D Burkart 1
Affiliation  

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.

中文翻译:


基于机制的交联探针捕获处于构象不同催化状态的大肠杆菌酮合酶 FabB



酮合成酶 (KS) 使用两步乒乓反应机制催化脂肪酸生物合成中必需的碳-碳键形成反应。在大肠杆菌中,有两种同型二聚体延伸KS,FabB和FabF,它们具有重叠的底物选择性。然而,FabB 对于在缺乏外源性 UFA 的情况下细胞生存所需的不饱和脂肪酸 (UFA) 的生物合成至关重要。此外,FabB 对长度超过 12 个碳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,解决了与 ACP 复合的 FabB 的两种交联晶体结构,并用近似催化步骤的长链脂肪酸交联探针进行功能化。两种同二聚体结构都具有不对称底物结合袋,表明两个 FabB 单体与 C14 和 C16 酰基链接合时存在合作关系。此外,这些结构捕获了活性位点门控残基 Phe392 的不寻常旋转异构体,它可能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。
更新日期:2022-09-01
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