Fernandes N D, Kolattukudy P E
Neurobiotechnology Center, The Ohio State University, Columbus, Ohio 43210, USA.
J Biol Chem. 1998 Jan 30;273(5):2823-8. doi: 10.1074/jbc.273.5.2823.
Mycobacterium bovis BCG produces a variety of methyl-branched fatty acids. They include C28 to C32 mycocerosic acids esterified to phthiocerol and phenolphthiocerol and the shorter (C22 to C26) mycocerosic acids esterified to phthiocerol. A mycocerosic acid synthase gene-disrupted mutant was still able to produce the shorter mycocerosic acids. The enzyme short chain mycocerosic acid synthase (SMAS), that catalyzes the synthesis of such acids, was purified using anion exchange and red-agarose chromatography. Gel filtration showed the native enzyme to be a 537-kDa protein. Since SDS-polyacrylamide gel electrophoresis of the purified enzyme showed a 280-, 170-, and 100-kDa protein and they cross-reacted with antibodies prepared against the 280- or 100-kDa protein, this enzyme is composed of the three subunits or two 280-kDa monomers with an unusual susceptibility to a proteolytic nick. SMAS utilizes methylmalonyl-CoA with C12 to C20 acyl-CoA as primers and with either NADH or NADPH as the reductant to synthesize the short mycocerosic acids. The Km values for NADH and NADPH were 93 and 90 microM, respectively. Antibodies raised against either the 280- or 100-kDa protein inhibited the incorporation of methylmalonyl-CoA into fatty acids by SMAS. The enzyme is not immunologically closely related to mycocerosic acid synthase or fatty acid synthase.
牛分枝杆菌卡介苗可产生多种甲基支链脂肪酸。它们包括与结核硬脂醇和酚结核硬脂醇酯化的C28至C32的霉菌酸，以及与结核硬脂醇酯化的较短的（C22至C26）霉菌酸。一个霉菌酸合酶基因破坏突变体仍能够产生较短的霉菌酸。催化此类酸合成的短链霉菌酸合酶（SMAS），通过阴离子交换和红琼脂糖色谱法进行了纯化。凝胶过滤显示天然酶是一种537 kDa的蛋白质。由于纯化酶的SDS-聚丙烯酰胺凝胶电泳显示出280 kDa、170 kDa和100 kDa的蛋白质，并且它们与针对280 kDa或100 kDa蛋白质制备的抗体发生交叉反应，因此该酶由三个亚基或两个280 kDa的单体组成，对蛋白水解切口具有异常的敏感性。SMAS利用甲基丙二酰辅酶A与C12至C20的酰基辅酶A作为引物，并以NADH或NADPH作为还原剂来合成短霉菌酸。NADH和NADPH的Km值分别为93和90 μM。针对280 kDa或100 kDa蛋白质产生的抗体抑制了SMAS将甲基丙二酰辅酶A掺入脂肪酸中。该酶在免疫学上与霉菌酸合酶或脂肪酸合酶没有密切关系。
