Identification of a novel NADH-specific aldo-keto reductase using sequence and structural homologies
Identification of a novel NADH-specific aldo-keto reductase using sequence and structural homologies
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DOI：
10.1042/bj20060660
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发表时间：
2006-11-15
影响因子：
4.1
通讯作者：
Wilson, David K.
中科院分区：
文献类型：
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作者：
Di Luccio, Eric;Elling, Robert A.;Wilson, David K.
醛酮还原酶（aldo-keto reductases，AKR）是一类主要依靠NADPH将各种含羰基化合物可逆还原为相应醇的酶超家族。少数已发现具有NADPH/NADH双重特异性，通常偏好NADPH，但没有一种是专门针对NADH的。双特异性酶木糖还原酶（AKR 2B 5）的晶体结构表明NAD+通过与谷氨酸的关键相互作用结合，谷氨酸能够改变构象以适应NADP+的2 '-磷酸。序列比较表明，类似的谷氨酸或天冬氨酸残基可能在其他AKRS中起作用，以允许利用NADH。在此基础上，从果蝇、大肠杆菌、粟酒裂殖酵母、硫磺硫化叶菌、苜蓿中华根瘤菌和海栖热袍菌中成功表达并纯化了7个基因。每一种都用常规AKR底物测定共底物依赖性。三个是NADPH的专属（AKR 2 E3，AKR 3F 2和AKR 3F 3），两个是双特异性的（AKR 3C 2和AKR 3F 1），一个是NADH的特异性（AKR 11B 2），这是AKR中的第一个这样的活性。第七种蛋白质的荧光测量表明，它结合NADPH和NADH，但没有活性。在NADH特异性大肠杆菌中天冬氨酸突变为丙氨酸残基或更易移动的谷氨酸。coli蛋白质将其转化为具有双重特异性的酶。这些结果表明，这种羧酸盐的存在是NADH依赖性的指示。这应该允许改进的预测的共底物特异性，并提供了一个基础，工程酶与改变共底物利用这类酶。
The AKRs (aldo-keto reductases) are a superfamily of enzymes which mainly rely on NADPH to reversibly reduce various carbonyl-containing compounds to the corresponding alcohols. A small number have been found with dual NADPH/NADH specificity, usually preferring NADPH, but none are exclusive for NADH. Crystal structures of the dual-specificity enzyme xylose reductase (AKR2B5) indicate that NAD+ is bound via a key interaction with a glutamate that is able to change conformations to accommodate the 2'-phosphate of NADP+. Sequence comparisons suggest that analogous glutamate or aspartate residues may function in other AKRS to allow NADH utilization. Based on this, nine putative enzymes with potential NADH specificity were identified and seven genes were successfully expressed and purified from Drosophila melanogaster, Escherichia coli, Schizosaccharomyces pombe, Sulfolobus solfataricus, Sinorhizobium meliloti and Thermotoga maritima. Each was assayed for co-substrate dependence with conventional AKR substrates. Three were exclusive for NADPH (AKR2E3, AKR3F2 and AKR3F3), two were dual-specific (AKR3C2 and AKR3F1) and one was specific for NADH (AKR11B2), the first such activity in an AKR. Fluorescence measurements of the seventh protein indicated that it bound both NADPH and NADH but had no activity. Mutation of the aspartate into an alanine residue or a more mobile glutamate in the NADH-specific E. coli protein converted it into an enzyme with dual specificity. These results show that the presence of this carboxylate is an indication of NADH dependence. This should allow improved prediction of co-substrate specificity and provide a basis for engineering enzymes with altered co-substrate utilization for this class of enzymes.