(127c) Cofactor Engineering of a Thermostable Aldo-Keto Reductase Enzyme: Altering Cofactor Specificity and Development of a Novel Directed Evolution Selection Platform

Aldo-keto reductases (AKRs) catalyze a wide range of reactions with a broad array of substrates, and are an attractive family of enzymes for use in biocatalysis applications including enzymatic biofuel cells. The cofactor binding pocket and active site are highly conserved within this superfamily, while substrate specificity is tailored through three substrate binding loops located above the active site. We will present our work on characterizing the cofactor binding mechanism of a thermostable AKR (an alcohol dehydrogenase) and the use of site directed mutagenesis to alter the cofactor specificity. We will also present a new method for the alteration of the cofactor specificity via directed evolution using a novel selection system. Additionally, we will discuss the extension of this system to allow modification of substrate specificity and the applications of this approach for the design of novel biocatalytic pathways.

Cofactor binding in the aldo-keto reductase superfamily has previously been investigated in several mammalian and prokaryotic AKR's. Here we report on an AKR from the hyperthermophilic archeon Pyrococcus furiosus, AdhD, which exhibits a different behavior from those AKR's already characterized. In contrast to the mesostable AKR's, the kinetic transient associated with cofactor binding does not seem to be dependent on the presence of the 2'-phosphate of NADP(H) or a consensus arginine in the binding pocket. Additionally, wild-type AdhD contains a histidine in place of the consensus arginine, which seems to be important in the enzyme's strong preference for NAD(H) over NADP(H), further distinguishing this AKR from most other family members. These differences were investigated through mutations to the cofactor binding pocket, where we attempted to rationally convert the cofactor specificity to NADP(H) and determine whether structure-function relationships established for other AKR's apply to this enzyme.

The extreme thermostability of this enzyme and the distinct cofactor and substrate binding pockets make it an attractive candidate for use in enzymatic biofuel cells and for general dehydrogenase applications. A novel selection scheme based on kinetic-based enzyme capture has been developed for use in directed evolution of desired cofactor and substrate specificities. This technique takes advantage of the ordered bi-bi reaction mechanism common to the AKR superfamily to select enzymes specific for the cofactor and substrate of interest. This may enable the creation of a family of enzymes with varying substrate and cofactor specificities which will benefit a variety of biotechnology applications.