Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity.
Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity.
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DOI：
10.1093/protein/gzv028
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发表时间：
2015-08
期刊：
影响因子：
--
通讯作者：
Gerth ML
中科院分区：
文献类型：
--
作者：
Maddock DJ;Patrick WM;Gerth ML
将酶的辅因子专一性从烟酰胺腺嘌呤二核苷酸2‘-磷酸(NADPH)改变为更丰富的NADH是微生物代谢工程中提高酶整体效率的常用策略。本研究的目的是将自产乙酸梭菌的初级-次级乙醇脱氢酶的辅因子特异性从严格依赖于NADPH转变为依赖于NADH。我们利用同源模型的见解建立了一个专注于残基S199的位置饱和文库，该位置被认为最有可能破坏NADPH的2‘-磷酸结合。虽然CaADH(S199X)文库没有产生任何依赖于NADH的酶，但它确实揭示了辅因子磷酸结合位点的替换可以导致酶底物专一性的意外变化。使用共识引导的定点突变，我们能够创造出一种严格依赖NADH的酶，尽管伴随着活性的降低。这项研究强调了末端残基在底物特异性和酶-辅因子相互作用的复杂性中所起的作用。
Changing the cofactor specificity of an enzyme from nicotinamide adenine dinucleotide 2′-phosphate (NADPH) to the more abundant NADH is a common strategy for increasing overall enzyme efficiency in microbial metabolic engineering. The aim of this study was to switch the cofactor specificity of the primary–secondary alcohol dehydrogenase from Clostridium autoethanogenum, a bacterium with considerable promise for the bio-manufacturing of fuels and other petrochemicals, from strictly NADPH-dependent to NADH-dependent. We used insights from a homology model to build a site-saturation library focussed on residue S199, the position deemed most likely to disrupt binding of the 2′-phosphate of NADPH. Although the CaADH(S199X) library did not yield any NADH-dependent enzymes, it did reveal that substitutions at the cofactor phosphate-binding site can cause unanticipated changes in the substrate specificity of the enzyme. Using consensus-guided site-directed mutagenesis, we were able to create an enzyme that was stringently NADH-dependent, albeit with a concomitant reduction in activity. This study highlights the role that distal residues play in substrate specificity and the complexity of enzyme–cofactor interactions.
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影响因子：
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