Biochemical characterization of acyl carrier protein (AcpM) and malonyl-CoA:AcpM transacylase (mtFabD), two major components of Mycobacterium tuberculosis fatty acid synthase II
Biochemical characterization of acyl carrier protein (AcpM) and malonyl-CoA:AcpM transacylase (mtFabD), two major components of Mycobacterium tuberculosis fatty acid synthase II
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DOI：
10.1074/jbc.m103687200
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发表时间：
2001-07-27
影响因子：
4.8
通讯作者：
Besra, GS
中科院分区：
文献类型：
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作者：
Kremer, L;Nampoothiri, KM;Besra, GS
丙二酰辅酶A（CoA）-酰基载体蛋白（ACP）转酰酶（MCAT）是包括结核分枝杆菌在内的所有细菌中脂肪酸生物合成的必需酶。MCAT催化丙二酸从丙二酰-CoA转酰为活化的holo-ACP，以产生丙二酰-ACP，其是脂肪酸生物合成中的延伸底物。为了阐明分枝杆菌酰基载体蛋白（AcpM）和MCAT在脂肪酸和分枝菌酸生物合成中的作用，我们从分枝杆菌中克隆、表达并纯化了acpM和mtfabD（malonyl-COA：AcpM transacylase）。结核根据所使用的培养条件，大肠杆菌中产生两种或三种不同形式的AcpM：apo-AcpM、holo-AcpM和棕榈酰化AcpM，正如电喷雾质谱所揭示的那样。mtfabD基因编码一个假定的MCAT被用来补充一个热敏E。colifabD突变体。mtFabD的表达和纯化导致在体外显示出强MCAT活性的活性酶。使用不同ACP底物的酶促研究确定了holo-AcpM构成mtFabD的优选底物。为了进一步了解mtFabD的结构-功能关系，产生了不同的突变蛋白。所有突变（Q9 A、R116 A、H194 A、Q243 A、S91 T和S91 A）在体外完全消除了MCAT活性，从而强调了这些残基在转酰化中的重要性。AcpM形式和mtFabD的产生和表征为在M中探索与脂肪酸和分枝菌酸生物合成相关的进一步研究开辟了道路。结核由于在分枝杆菌中发现了一种特定类型的FabD，因此它代表了一种等待开发的有吸引力的新药物靶标。
Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in the biosynthesis of fatty acids in all bacteria, including Mycobacterium tuberculosis. MCAT catalyzes the transacylation of malonate from malonyl-CoA to activated holo-ACP, to generate malonyl-ACP, which is an elongation substrate in fatty acid biosynthesis. To clarify the roles of the mycobacterial acyl carrier protein (AcpM) and MCAT in fatty acid and mycolic acid biosynthesis, we have cloned, expressed, and purified acpM and mtfabD (malonyl-COA: AcpM transacylase) from M. tuberculosis. According to the culture conditions used, AcpM was produced in Escherichia coli in two or three different forms: apo-AcpM, holo-AcpM, and palmitoylated-AcpM, as revealed by electrospray mass spectrometry. The mtfabD gene encoding a putative MCAT was used to complement a thermosensitive E. coli fabD mutant. Expression and purification of mtFabD resulted in an active enzyme displaying strong MCAT activity in vitro. Enzymatic studies using different ACP substrates established that holo-AcpM constitutes the preferred substrate for mtFabD. In order to provide further insight into the structure-function relationship of mtFabD, different mutant proteins were generated. All mutations (Q9A, R116A, H194A, Q243A, S91T, and S91A) completely abrogated MCAT activity in vitro, thus underlining the importance of these residues in transacylation. The generation and characterization of the AcpM forms and mtFabD opens the way for further studies relating to fatty acid and mycolic acid biosynthesis to be explored in M. tuberculosis. Since a specific type of FabD is found in mycobacterial species, it represents an attractive new drug target waiting to be exploited.