The malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) plays a key role in cell wall biosynthesis in Mycobacterium tuberculosis and other bacteria. The M. tuberculosis MCAT (MtMCAT) is encoded by the FabD gene and catalyzes the transacylation of malonate from malonyl-CoA to holo-ACP. Malonyl-ACP is the substrate in fatty acid biosynthesis and is a by-product of the transacylation reaction. This ability for fatty acid biosynthesis enables M. tuberculosis to survive in hostile environments, and thus understanding the mechanism of biosynthesis is important for the design of new anti-tuberculosis drugs. The 2.3 A crystal structure of MtMCAT reported here shows that its catalytic mechanism differs from those of ScMCAT and EcMCAT, whose structures have previously been determined. In MtMCAT, the C(beta)-O(gamma) bond of Ser91 turns upwards, resulting in a different orientation and thus an overall change of the active pocket compared to other known MCAT enzymes. We identify three new nucleophilic attack chains from the MtMCAT structure: His90-Ser91, Asn155-Wat6-Ser91 and Asn155-His90-Ser91. Enzyme activity assays show that His90A, Asn155A and His90A-Asn155A mutants all have substantially reduced MCAT activity, indicating that M. tuberculosis MCAT supports a unique means of proton transfer. Furthermore, His194 cannot form part of a His-Ser catalytic dyad and only stabilizes the substrate. This new discovery should provide a deeper insight into the catalytic mechanisms of MCATs.

译文

丙二酰辅酶a (CoA)-酰基载体蛋白 (ACP) 转酰基酶 (MCAT) 在结核分枝杆菌和其他细菌的细胞壁生物合成中起关键作用。结核分枝杆菌MCAT (MtMCAT) 由FabD基因编码,可催化丙二酸从丙二酰辅酶a转酰化为holo-ACP。丙二酰-ACP是脂肪酸生物合成中的底物,是转酰化反应的副产物。这种脂肪酸生物合成的能力使结核分枝杆菌能够在恶劣的环境中生存,因此了解生物合成的机理对于设计新的抗结核药物很重要。本文报道的MtMCAT的2.3 A晶体结构表明其催化机理不同于ScMCAT和EcMCAT的催化机理,ScMCAT和EcMCAT的结构先前已经确定。在MtMCAT中,与其他已知的MCAT酶相比,Ser91的C (β)-O (γ) 键向上旋转,导致不同的方向,从而导致活性口袋的整体变化。我们从MtMCAT结构中识别出三个新的亲核攻击链: His90-Ser91,Asn155-Wat6-Ser91和Asn155-His90-Ser91。酶活性测定表明His90A,Asn155A和His90A-Asn155A突变体均具有显着降低的MCAT活性,表明结核分枝杆菌MCAT支持质子转移的独特手段。此外,His194不能形成His-Ser催化二联体的一部分,只能稳定底物。这一新发现将为MCATs的催化机制提供更深入的了解。

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