Probing the mechanism of the Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein synthase III mtFabH -: Factors influencing catalysis and substrate specificity
Probing the mechanism of the Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein synthase III mtFabH -: Factors influencing catalysis and substrate specificity
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DOI：
10.1074/jbc.m413216200
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发表时间：
2005-09-16
影响因子：
4.8
通讯作者：
Besra, GS
中科院分区：
文献类型：
--
作者：
Brown, AK;Sridharan, S;Besra, GS
霉菌酸是结核分枝杆菌细胞壁的主要特征。这些-烷基-羟基脂肪酸是由两种脂肪酸缩合而成，一种是长meromycolic酸，一种是短C-24-C-26脂肪酸。组成脂肪酸是通过I型和II型脂肪酸合成酶（FAS）的组合产生的，FAS-I产物被FAS-II拉长成meromycolic酸。mtfabH编码的β -酮酰基酰基载体蛋白（ACP）合成酶III连接FAS-I和FAS-II，催化FAS-I衍生的酰基辅酶a与丙二酰酰基载体蛋白（ACP）缩合。体外评估mtFabH的酰基辅酶a链长特异性；与天然伴侣AcpM配对时，酶延长了与生理相关的酰基辅酶a引物，而不是与大肠杆菌ACP配对。该酶利用大肠杆菌ACP的能力表明，与这两种ACP的结合模式可能相似，但很明显，AcpM固有的独特因素调节了mtFabH的底物特异性。提出的关键mtFabH残基的突变被用来定义它们的催化作用。假定的酰基辅酶a结合残基的取代减少了转酰基化，双取代完全废除了活性。Arg（46）的突变表明，它在丙二酰- acpm脱羧中的作用比在酰基- coa结合中的作用更为关键。有趣的是，这种效应被Arg(161) -> α取代所抑制。我们的结构研究表明，先前涉及丙二酰acp脱羧的His（258）也作为水分子网络的锚点，我们提出促进Cys的去质子化和转酰基化（122）。
Mycolic acids are the dominant feature of the Mycobacterium tuberculosis cell wall. These alpha-alkyl, beta-hydroxy fatty acids are formed by the condensation of two fatty acids, a long meromycolic acid and a shorter C-24-C-26 fatty acid. The component fatty acids are produced via a combination of type I and II fatty acid synthases (FAS) with FAS-I products being elongated by FAS-II toward meromycolic acids. The beta-ketoacyl-acyl carrier protein (ACP) synthase III encoded by mtfabH (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-acyl carrier protein ( ACP). The acyl-CoA chain length specificity of mtFabH was assessed in vitro; the enzyme extended longer, physiologically relevant acyl-CoA primers when paired with AcpM, its natural partner, than with Escherichia coli ACP. The ability of the enzyme to use E. coli ACP suggests that a similar mode of binding is likely with both ACPs, yet it is clear that unique factors inherent to AcpM modulate the substrate specificity of mtFabH. Mutation of proposed key mtFabH residues was used to define their catalytic roles. Substitution of supposed acyl-CoA binding residues reduced transacylation, with double substitutions totally abrogating activity. Mutation of Arg(46) revealed its more critical role in malonyl-AcpM decarboxylation than in the acyl-CoA binding role. Interestingly, this effect was suppressed intragenically by Arg(161) -> Ala substitution. Our structural studies suggested that His(258), previously implicated in malonyl-ACP decarboxylation, also acts as an anchor point for a network of water molecules that we propose promotes deprotonation and transacylation of Cys(122).