Probing reactivity and substrate specificity of both subunits of the dimeric Mycobacterium tuberculosis FabH using alkyl-CoA disulfide inhibitors and acyl-CoA substrates.
Probing reactivity and substrate specificity of both subunits of the dimeric Mycobacterium tuberculosis FabH using alkyl-CoA disulfide inhibitors and acyl-CoA substrates.
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使用烷基辅酶 A 二硫化物抑制剂和酰基辅酶 A 底物探测二聚体结核分枝杆菌 FabH 两个亚基的反应性和底物特异性。
DOI：
10.1016/j.bioorg.2007.11.001
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发表时间：
2008
影响因子：
5.1
通讯作者：
Reynolds,KevinA
中科院分区：
文献类型：
--
作者：
Sachdeva,Sarbjot;Musayev,Faik;Alhamadsheh,MamounM;NeelScarsdale,J;TonieWright,H;Reynolds,KevinA
二聚体结核分枝杆菌FabH （mtFabH）催化酰基辅酶a和丙二酰酰基载体蛋白（ACP）之间的claisen型缩合，启动II型脂肪酸合成酶循环。为了分析mtFabH与酰基辅酶a的初始共价酰化，我们用c6 - c20酰基辅酶a的混合物对其进行了挑战，ESI-MS分析显示两个亚基都有反应，并且对c12酰基辅酶a有严格的特异性。用十烷基辅酶a二硫抑制剂对mtFabH进行的晶体学和ESI-MS研究显示，通过与活性位点半胱氨酸的二硫键连接，两个亚基的酰基结合通道中都有一个十烷基链。这些数据提供了第一个明确的证据，证明mtFabH的两个亚基都可以与底物或抑制剂反应。在初始酰化步骤中观察到的C12acyl-CoA底物特异性与mtFabH在整个mtFabH催化反应中对C18-C20acyl-CoA底物的更高催化效率之间的差异表明结核分枝杆菌ACP在该反应中起着特异性决定因素的作用。
The dimeric Mycobacterium tuberculosis FabH (mtFabH) catalyses a Claisen-type condensation between an acyl-CoA and malonyl-acyl carrier protein (ACP) to initiate the Type II fatty acid synthase cycle. To analyze the initial covalent acylation of mtFabH with acyl-CoA, we challenged it with mixture of C6-C20acyl-CoAs and the ESI-MS analysis showed reaction at both subunits and a strict specificity for C12acyl CoA. Crystallographic and ESI-MS studies of mtFabH with a decyl-CoA disulfide inhibitor revealed a decyl chain bound in acyl-binding channels of both subunits through disulfide linkage to the active site cysteine. These data provide the first unequivocal evidence that both subunits of mtFabH can react with substrates or inhibitor. The discrepancy between the observed C12acyl-CoA substrate specificity in the initial acylation step and the higher catalytic efficiency of mtFabH for C18-C20acyl-CoA substrates in the overall mtFabH catalyzed reaction suggests a role for M. tuberculosis ACP as a specificity determinant in this reaction.