Lee Sungwon, Poulter C Dale
Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112, USA.
J Bacteriol. 2008 Jun;190(11):3808-16. doi: 10.1128/JB.01939-07. Epub 2008 Mar 28.
Squalene synthase (SQS) is a bifunctional enzyme that catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to give presqualene diphosphate (PSPP) and the subsequent rearrangement of PSPP to squalene. These reactions constitute the first pathway-specific steps in hopane biosynthesis in Bacteria and sterol biosynthesis in Eukarya. The genes encoding SQS were isolated from the hopane-producing bacteria Thermosynechococcus elongatus BP-1, Bradyrhizobium japonicum, and Zymomonas mobilis and cloned into an Escherichia coli expression system. The expressed proteins with a His(6) tag were found exclusively in inclusion bodies when no additives were used in the buffer. After extensive optimization, soluble recombinant T. elongatus BP-1 SQS was obtained when cells were disrupted and purified in buffers containing glycerol. The recombinant B. japonicum and Z. mobilis SQSs could not be solubilized under any of the expression and purification conditions used. Purified T. elongatus His(6)-SQS gave a single band at 42 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular ion at m/z 41886 by electrospray mass spectrometry. Incubation with FPP and NADPH gave squalene as the sole product. Incubation of the enzyme with [(14)C]FPP in the absence of NADPH gave PSPP. The enzyme requires Mg(2+) for activity, has an optimum pH of 7.6, and is strongly stimulated by detergent. Under optimal conditions, the K(m) of FPP is 0.97 +/- 0.10 microM and the k(cat) is 1.74 +/- 0.04 s(-1). Zaragozic acid A, a potent inhibitor of mammalian, fungal, and Saccharomyces cerevisiae SQSs, also inhibited recombinant T. elongatus BP-1 SQS, with a 50% inhibitory concentration of 95.5 +/- 13.6 nM.
角鲨烯合酶（SQS）是一种双功能酶，它催化两分子法呢基二磷酸（FPP）缩合生成前角鲨烯二磷酸（PSPP），随后PSPP重排为角鲨烯。这些反应是细菌中藿烷生物合成和真核生物中甾醇生物合成途径中特定的第一步。编码SQS的基因从产藿烷的细菌嗜热栖热菌BP - 1、日本慢生根瘤菌和运动发酵单胞菌中分离出来，并克隆到大肠杆菌表达系统中。当缓冲液中不使用添加剂时，带有His(6)标签的表达蛋白仅存在于包涵体中。经过广泛优化，当细胞在含有甘油的缓冲液中破碎和纯化时，获得了可溶性重组嗜热栖热菌BP - 1 SQS。在所用的任何表达和纯化条件下，重组日本慢生根瘤菌和运动发酵单胞菌SQS都不能溶解。纯化的嗜热栖热菌His(6)-SQS经十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳在42 kDa处出现单一条带，电喷雾质谱分析给出的分子离子峰为m/z 41886。与FPP和NADPH一起温育时，角鲨烯是唯一产物。在没有NADPH的情况下，该酶与[(14)C]FPP温育时产生PSPP。该酶活性需要Mg(2+)，最适pH为7.6，并且受到去污剂的强烈刺激。在最佳条件下，FPP的K(m)为0.97±0.10 microM，k(cat)为1.74±0.04 s(-1)。扎拉戈昔酸A是哺乳动物、真菌和酿酒酵母SQS的有效抑制剂，也抑制重组嗜热栖热菌BP - 1 SQS，其50%抑制浓度为95.5±13.6 nM。
