Altering the Substrate and Cofactor Specificities of a Dehydrogenase
Dehydrogenase enzymes are commonly used in a wide range of biotechnology applications, including chemical synthesis and bioelectrocatalysis. Aldo-keto reductases (AKRs) are a family of enzymes that catalyze a wide range of redox reactions with a broad array of substrates, and are an attractive family of enzymes for protein engineering efforts. They are monomeric and they fold into the well-known TIM-like alpha/beta structure. The cofactor binding pocket and the active site are highly conserved within the superfamily, while substrate specificity is tailored through three substrate binding loops located above the active site.
We have rationally altered the cofactor specificity of a bacterial AKR, the 2,5-diketo-D-gluconic acid reductase (2,5-DKGR) which is an important enzyme for the enzymatic production of vitamin C. Both site-directed mutations as well as combinatorial approaches were used. The native enzyme is strongly NADP(H)-dependent and through this protein engineering approach a new mutant was created that retained its NADP(H) activity while gaining activity with NAD(H) that is close to the wild type activity with NADP(H).
More recently we have been exploring the cofactor and substrate specificity of the AhdD enzyme which is an AKR from the hyperthermophilic archeon Pyrococcus furiosus. This enzyme natively uses NAD(H) as its preferred cofactor and the mutations used to alter cofactor specificity in 2,5-DKGR were used to similarly broaden the cofactor specificity of AdhD. The mutant AdhD is also able to use biomimetic cofactors, and we have used this mutant to create an enzymatic biofuel cell using an biomimetic cofactor for redox cycling. We have also explored swapping the substrate-binding loops in AdhD to alter its substrate specificity in addition to its cofactor specificity. AdhD is a valuable enzymatic scaffold for engineering enzymes with high thermostability.
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