Takayama Kuni, Hayes Barry, Vestling Matha M, Massey Randall J
Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine, Madison, WI 53706, USA.
Biochem J. 2003 Jul 15;373(Pt 2):465-74. doi: 10.1042/BJ20030248.
Enzymes within the biosynthetic pathway of mycolic acid (C(60)-C(90) a-alkyl,b-hydroxyl fatty acid) in Mycobacterium tuberculosis are attractive targets for developing new anti-tuberculosis drugs. We have turned to the simple model system of Corynebacterium matruchotii to study the terminal steps in the anabolic pathway of a C32 mycolic acid called corynomycolic acid. By transposon-5 mutagenesis, we transformed C. matruchotii into a mutant that is unable to synthesize corynomycolic acid. Instead, it synthesized two related series of novel fatty acids that were released by saponification from the cell wall fraction and from two chloroform/methanol-extractable glycolipids presumed to be analogues of trehalose mono- and di-corynomycolate. By chemical analyses and MS, we determined the general structure of the two series to be 2,4,6,8,10-penta-alkyl decanoic acid for the larger series (C(70)-C(77)) and 2,4,6,8-tetra-alkyl octanoic acid for the smaller series (C(52)-C(64)), both containing multiple keto groups, hydroxy groups and double bonds. The mutant was temperature-sensitive, aggregated extensively, grew very slowly relative to the wild type, and was resistant to the presence of lysozyme. We suggest that a regulatory protein that normally prevents the transfer of the condensation product back to b-ketoacyl synthase in the corynomycolate synthase system of the wild type was inactivated in the mutant. This will result in multiple Claisen-type condensation and the formation of two similar series of these complex hybrid fatty acids. A similar protein in M. tuberculosis would be an attractive target for new drug discovery.
结核分枝杆菌中分枝菌酸（C(60)-C(90) α-烷基，β-羟基脂肪酸）生物合成途径中的酶是开发新型抗结核药物的有吸引力的靶点。我们转向了简单的马氏棒状杆菌模型系统，以研究一种名为棒状分枝菌酸的C32分枝菌酸合成途径中的末端步骤。通过转座子5诱变，我们将马氏棒状杆菌转化为一种无法合成棒状分枝菌酸的突变体。相反，它合成了两个相关的新型脂肪酸系列，这些脂肪酸通过皂化从细胞壁部分以及从两种假定为海藻糖单棒状分枝菌酸酯和二棒状分枝菌酸酯类似物的氯仿/甲醇可提取糖脂中释放出来。通过化学分析和质谱，我们确定这两个系列的一般结构为较大系列（C(70)-C(77)）的2,4,6,8,10-五烷基癸酸和较小系列（C(52)-C(64)）的2,4,6,8-四烷基辛酸，两者都含有多个酮基、羟基和双键。该突变体对温度敏感，大量聚集，相对于野生型生长非常缓慢，并且对溶菌酶的存在具有抗性。我们认为，一种通常在野生型棒状分枝菌酸合酶系统中阻止缩合产物回传给β-酮酰基合酶的调节蛋白在突变体中失活。这将导致多次克莱森型缩合，并形成两个类似系列的这些复杂杂合脂肪酸。结核分枝杆菌中的类似蛋白将是新药发现的有吸引力的靶点。
