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Extreme makeover: Engineering the activity of a thermostable alcohol dehydrogenase (AdhD) from Pyrococcus furiosus
Author(s) -
Solanki Kusum,
Abdallah Walaa,
Banta Scott
Publication year - 2016
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201600152
Subject(s) - cofactor , pyrococcus furiosus , protein engineering , biochemistry , directed evolution , alcohol dehydrogenase , active site , enzyme , chemistry , dehydrogenase , oxidoreductase , substrate (aquarium) , nicotinamide mononucleotide , nad+ kinase , biology , mutant , archaea , ecology , gene , nicotinamide adenine dinucleotide
Abstract 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.