(439g) Protein Engineering of a Thermostable Alcohol Dehydrogenase to Improve Activity with Biomimetic Cofactors and Alternate Substrates

Dehydrogenase enzymes are commonly used in a wide range of biotechnology applications, including cofactor regeneration and bioelectrocatalysis. However, the use of wild-type dehydrogenases is often limited by cofactor requirements, substrate specificity, and stability in immobilized architectures. Thus we are interested in developing a general dehydrogenase platform that addresses these limitations and is optimized for creating bioelectrode modifications for use in biosensors and biofuel cells. To this end, we have engineered a thermostable dehydrogenase scaffold capable of self-assembly, broadened the cofactor specificity of the enzyme allowing for the use of minimal cofactors, and are currently developing a novel directed evolution selection step to allow rapid alteration of substrate specificity.

A thermostable alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus was selected for this work due to its extreme stability and broad substrate specificity. The enzyme, AdhD, belongs to the well characterized aldo-keto reductase (AKR) superfamily, and has a strong preference for NAD(H) and the oxidation of secondary diols. Based on previous work elucidating the determinants of cofactor specificity in AKR's, two rational mutations were made to the cofactor binding pocket of AdhD. The resulting K249G/H255R double mutant exhibited an up to 150-fold improvement in activity and broadened cofactor specificity compared to the wild-type enzyme, while thermal stability was unaffected. Additionally, the mutant enzyme demonstrated activity with the minimal cofactor nicotinamide mononucleotide (NMN) both in kinetic assays and when immobilized as a bioanode. As cofactor diffusion has been shown to be rate-limiting in some immobilized enzyme architectures, the use of minimal cofactors could significantly improve performance in these applications.

In order to improve the activity of the enzyme with glucose, substrate binding loops were grafted into the AdhD scaffold from human aldose reductase (hAR), a mesostable aldo-keto reductase. Substitution of the three hAR loops into the wild-type AdhD altered both cofactor and substrate specificity, and allowed AdhD to reduce DL-glyceraldehyde using NADPH. Interestingly, when these loops were grafted into the double mutant that had previously demonstrated improved activity with NADPH, no DL-glyceraldehyde reduction was observed.

Additionally, we are developing and optimizing a directed evolution selection system, based on the kinetic-based enzyme capture technique, for identifying enzymes with desired substrate specificities. This approach takes advantage of the ordered bi-bi kinetic mechanism followed by the aldo-keto reductases to separate enzymes with the desired cofactor and substrate specificity. The ability to engineer novel specificities into the designed dehydrogenase scaffold will be a powerful protein engineering tool, and we envision using this approach to create a multi-step enzymatic biofuel cell capable of the serial oxidation of biofuels.