Solanki Kusum, Abdallah Walaa, Banta Scott
Department of Chemical Engineering, Columbia University in the City of New York, New York, NY, USA.
Biotechnol J. 2016 Dec;11(12):1483-1497. doi: 10.1002/biot.201600152. Epub 2016 Sep 5.
Alcohol dehydrogenase D (AdhD) is a monomeric thermostable alcohol dehydrogenase from the aldo-keto reductase (AKR) superfamily of proteins. We have been exploring various strategies of engineering the activity of AdhD so that it could be employed in future biotechnology applications. Driven by insights made in other AKRs, we have made mutations in the cofactor-binding pocket of the enzyme and broadened its cofactor specificity. A pre-steady state kinetic analysis yielded new insights into the conformational behavior of this enzyme. The most active mutant enzyme concomitantly gained activity with a non-native cofactor, nicotinamide mononucleotide, NMN(H), and an enzymatic biofuel cell was demonstrated with this enzyme/cofactor pair. Substrate specificity was altered by grafting loop regions near the active site pocket from a mesostable human aldose reductase (hAR) onto the thermostable AdhD. These moves not only transferred the substrate specificity of hAR but also the cofactor specificity of hAR. We have added alpha-helical appendages to AdhD to enable it to self-assemble into a thermostable catalytic proteinaceous hydrogel. As our understanding of the structure/function relationship in AdhD and other AKRs advances, this ubiquitous protein scaffold could be engineered for a variety of catalytic activities that will be useful for many future applications.
乙醇脱氢酶D（AdhD）是一种来自醛酮还原酶（AKR）超家族蛋白质的单体热稳定乙醇脱氢酶。我们一直在探索各种工程化AdhD活性的策略，以便它能在未来的生物技术应用中得到应用。受其他AKR研究成果的启发，我们对该酶的辅因子结合口袋进行了突变，拓宽了其辅因子特异性。预稳态动力学分析为该酶的构象行为提供了新的见解。活性最高的突变酶同时获得了对非天然辅因子烟酰胺单核苷酸NMN(H)的活性，并且用这种酶/辅因子对展示了一个酶促生物燃料电池。通过将中稳定性人醛糖还原酶（hAR）活性位点口袋附近的环区域嫁接到热稳定的AdhD上，改变了底物特异性。这些举措不仅转移了hAR的底物特异性，还转移了hAR的辅因子特异性。我们在AdhD上添加了α-螺旋附属物，使其能够自组装成一种热稳定的催化蛋白质水凝胶。随着我们对AdhD和其他AKR中结构/功能关系的理解不断深入，这种普遍存在的蛋白质支架可以被设计用于多种催化活性，这将对未来的许多应用有用。
