Wallace K K, Bao Z Y, Dai H, Digate R, Schuler G, Speedie M K, Reynolds K A
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland at Baltimore MD, USA.
Eur J Biochem. 1995 Nov 1;233(3):954-62. doi: 10.1111/j.1432-1033.1995.954_3.x.
A crotonyl-CoA reductase (EC 1.3.1.38, acyl-CoA:NADP+ trans-2-oxidoreductase) catalyzing the conversion of crotonyl-CoA to butyryl-CoA has been purified and characterized from Streptomyces collinus. This enzyme, a dimer with subunits of identical mass (48 kDa), exhibits a Km = 18 microM for crotonyl-CoA and 15 microM for NADPH. The enzyme was unable to catalyze the reduction of any other enoyl-CoA thioesters or to utilize NADH as an electron donor. A highly effective inhibition by straight-chain fatty acids (Ki = 9.5 microM for palmitoyl-CoA) compared with branched-chain fatty acids (Ki > 400 microM for isopalmitoyl-CoA) was observed. All of these properties are consistent with a proposed role of the enzyme in providing butyryl-CoA as a starter unit for straight-chain fatty acid biosynthesis. The crotonyl-CoA reductase gene was cloned in Escherichia coli. This gene, with a proposed designation of ccr, is encoded in a 1344-bp open reading frame which predicts a primary translation product of 448 amino acids with a calculated molecular mass of 49.4 kDa. Several dispersed regions of highly significant sequence similarity were noted between the deduced amino acid sequence and various alcohol dehydrogenases and fatty acid synthases, including one region that contains a putative NADPH binding site. The ccr gene product was expressed in E. coli and the induced crotonyl-CoA reductase was purified tenfold and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate/polyacrylamide to the native protein.
已从委内瑞拉链霉菌中纯化并鉴定了一种巴豆酰辅酶A还原酶（EC 1.3.1.38，酰基辅酶A：NADP⁺反式-2-氧化还原酶），该酶催化巴豆酰辅酶A转化为丁酰辅酶A。这种酶是一种由相同质量亚基（48 kDa）组成的二聚体，对巴豆酰辅酶A的Km值为18 μM，对NADPH的Km值为15 μM。该酶无法催化任何其他烯酰辅酶A硫酯的还原反应，也不能利用NADH作为电子供体。观察到直链脂肪酸对其有高效抑制作用（棕榈酰辅酶A的Ki = 9.5 μM），而支链脂肪酸的抑制作用较弱（异棕榈酰辅酶A的Ki > 400 μM）。所有这些特性都与该酶在为直链脂肪酸生物合成提供丁酰辅酶A起始单元中所起的作用相一致。巴豆酰辅酶A还原酶基因已克隆到大肠杆菌中。该基因暂定名为ccr，由一个1344 bp的开放阅读框编码，预测其初级翻译产物为448个氨基酸，计算分子量为49.4 kDa。在推导的氨基酸序列与各种醇脱氢酶和脂肪酸合酶之间发现了几个高度显著的序列相似性分散区域，其中一个区域包含一个假定的NADPH结合位点。ccr基因产物在大肠杆菌中表达，诱导表达的巴豆酰辅酶A还原酶经纯化后活性提高了10倍，并且在十二烷基硫酸钠/聚丙烯酰胺凝胶上显示出与天然蛋白相似的稳态动力学和电泳迁移率。
