Mechanistic details of the actinobacterial lyase-catalyzed degradation reaction of 2-hydroxyisobutyryl-CoA.
Mechanistic details of the actinobacterial lyase-catalyzed degradation reaction of 2-hydroxyisobutyryl-CoA.
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放线菌裂解酶催化 2-羟基异丁酰-CoA 降解反应的机制细节。
DOI：
10.1016/j.jbc.2021.101522
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发表时间：
2022-01
期刊：
影响因子：
--
通讯作者：
Rohwerder T
中科院分区：
文献类型：
--
作者：
Zahn M;König G;Pham HVC;Seroka B;Lazny R;Yang G;Ouerfelli O;Lotowski Z;Rohwerder T
放线菌2-羟酰-CoA裂解酶可逆地催化2-羟异丁酰-CoA的二磷酸硫胺素依赖性裂解为甲酰基-CoA和丙酮。这种酶在化学品的可持续生产的合成一碳同化途径中具有很大的潜力，但缺乏底物结合和生化重组反应机制的细节。我们确定了四聚体酶的晶体结构在封闭的构象与结合底物，共价postcleavage中间体，和产品，发光活性位点的架构和底物的相互作用。结合共价裂解前复合物的分子动力学模拟，完整的催化循环在结构上被描绘出来，揭示了从底物酰基Cβ羟基到残基E493的质子转移，随后将其返回到裂解后的Cα-碳负离子中间体。突变体E493 A、E493 Q和E493 K获得的动力学参数证实了E493在WT酶中的催化作用。然而，与WT相比，E493 A和E493 Q突变体中裂解酶活性分别降低10倍和50倍，表明水分子可能有助于质子转移。推定的催化谷氨酸位于靠近活性位点的短α-螺旋上。这种结构特征似乎在相关裂解酶中是保守的，例如人2-羟酰基-CoA裂解酶2。有趣的是，放线菌2-羟酰基-CoA裂解酶的一个独特特征是一个大的C-末端盖结构域，与活性位点残基L127和I492一起，将底物大小限制为≤C5 2-羟酰基残基。这些细节的催化机制和底物特异性的决定因素铺平了道路，为设计量身定制的催化剂，用于生物技术应用中的一个碳和短链底物的偶姻缩合。
Actinobacterial 2-hydroxyacyl-CoA lyase reversibly catalyzes the thiamine diphosphate-dependent cleavage of 2-hydroxyisobutyryl-CoA to formyl-CoA and acetone. This enzyme has great potential for use in synthetic one-carbon assimilation pathways for sustainable production of chemicals, but lacks details of substrate binding and reaction mechanism for biochemical reengineering. We determined crystal structures of the tetrameric enzyme in the closed conformation with bound substrate, covalent postcleavage intermediate, and products, shedding light on active site architecture and substrate interactions. Together with molecular dynamics simulations of the covalent precleavage complex, the complete catalytic cycle is structurally portrayed, revealing a proton transfer from the substrate acyl Cβ hydroxyl to residue E493 that returns it subsequently to the postcleavage Cα-carbanion intermediate. Kinetic parameters obtained for mutants E493A, E493Q, and E493K confirm the catalytic role of E493 in the WT enzyme. However, the 10- and 50-fold reduction in lyase activity in the E493A and E493Q mutants, respectively, compared with WT suggests that water molecules may contribute to proton transfer. The putative catalytic glutamate is located on a short α-helix close to the active site. This structural feature appears to be conserved in related lyases, such as human 2-hydroxyacyl-CoA lyase 2. Interestingly, a unique feature of the actinobacterial 2-hydroxyacyl-CoA lyase is a large C-terminal lid domain that, together with active site residues L127 and I492, restricts substrate size to ≤C5 2-hydroxyacyl residues. These details about the catalytic mechanism and determinants of substrate specificity pave the ground for designing tailored catalysts for acyloin condensations for one-carbon and short-chain substrates in biotechnological applications.
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影响因子：
64.8
作者：
Jumper J;Evans R;Pritzel A;Green T;Figurnov M;Ronneberger O;Tunyasuvunakool K;Bates R;Žídek A;Potapenko A;Bridgland A;Meyer C;Kohl SAA;Ballard AJ;Cowie A;Romera-Paredes B;Nikolov S;Jain R;Adler J;Back T;Petersen S;Reiman D;Clancy E;Zielinski M;Steinegger M;Pacholska M;Berghammer T;Bodenstein S;Silver D;Vinyals O;Senior AW;Kavukcuoglu K;Kohli P;Hassabis D
通讯作者：
Hassabis D
影响因子：
56.9
作者：
Frank, RAW;Titman, CM;Perham, RN
通讯作者：
Perham, RN
影响因子：
14.8
作者：
Dai, Lunzhi;Peng, Chao;Zhao, Yingming
通讯作者：
Zhao, Yingming
影响因子：
3.2
作者：
BAETZ, AL;ALLISON, MJ
通讯作者：
ALLISON, MJ
影响因子：
64.8
作者：
Dai, Shaobo;Funk, Lisa-Marie;Tittmann, Kai
通讯作者：
Tittmann, Kai