Department of Bioengineering, College of Life Science, Dalian Minzu University, Dalian 116600, China.
Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, College of Life Science, Dalian Minzu University, Dalian 116600, China.
Int J Mol Sci. 2023 Feb 3;24(3):2994. doi: 10.3390/ijms24032994.
Butanol dehydrogenase (BDH) plays a significant role in the biosynthesis of butanol in bacteria by catalyzing butanal conversion to butanol at the expense of the NAD(P)H cofactor. BDH is an attractive enzyme for industrial application in butanol production; however, its molecular function remains largely uncharacterized. In this study, we found that YqdH (FnYqdH) converts aldehyde into alcohol by utilizing NAD(P)H, with broad substrate specificity toward aldehydes but not alcohols. An in vitro metal ion substitution experiment showed that FnYqdH has higher enzyme activity in the presence of Co. Crystal structures of FnYqdH, in its apo and complexed forms (with NAD and Co), were determined at 1.98 and 2.72 Å resolution, respectively. The crystal structure of apo- and cofactor-binding states of FnYqdH showed an open conformation between the nucleotide binding and catalytic domain. Key residues involved in the catalytic and cofactor-binding sites of FnYqdH were identified by mutagenesis and microscale thermophoresis assays. The structural conformation and preferred optimal metal ion of FnYqdH differed from that of TmBDH (homolog protein of FnYqdH). Overall, we proposed an alternative model for putative proton relay in FnYqdH, thereby providing better insight into the molecular function of BDH.
丁醇脱氢酶 (BDH) 通过催化丁醛转化为丁醇，消耗 NAD(P)H 辅因子，在细菌中丁醇的生物合成中发挥重要作用。BDH 是工业生产丁醇中一种有吸引力的酶；然而，其分子功能在很大程度上仍未得到充分表征。在这项研究中，我们发现 YqdH（FnYqdH）利用 NAD(P)H 将醛转化为醇，对醛具有广泛的底物特异性，但对醇没有特异性。体外金属离子替代实验表明，FnYqdH 在存在 Co 的情况下具有更高的酶活性。FnYqdH 的apo 和复合物形式（与 NAD 和 Co 结合）的晶体结构分别在 1.98 和 2.72 Å 分辨率下确定。apo 和辅因子结合状态的 FnYqdH 的晶体结构显示核苷酸结合和催化结构域之间呈开放构象。通过突变和微量热泳动测定鉴定了 FnYqdH 的催化和辅因子结合位点中的关键残基。FnYqdH 的结构构象和优选的最佳金属离子与 TmBDH（FnYqdH 的同源蛋白）不同。总体而言，我们提出了 FnYqdH 中假定质子传递的替代模型，从而更好地了解 BDH 的分子功能。
