There have been many reports exploring the engineering of the cofactor specificity of aldo-keto reductases (AKRs), as this class of proteins is ubiquitous and exhibits many useful activities. A common approach is the mutagenesis of amino acids involved in interactions with the 2′-phosphate group of NADP(H) in the cofactor binding pocket. We recently performed a ‘loop-grafting’ approach to engineer the substrate specificity of the thermostable alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus and we found that a loop insertion after residue 211, which is on the back side of the cofactor binding pocket, could also alter cofactor specificity. Here, we further explore this approach by introducing single point mutations and single amino acid insertions at the loop insertion site. Six different mutants of AdhD were created by either converting glycine 211 to cysteine or serine or by inserting alanine, serine, glycine or cysteine between the 211 and 212 residues. Several mutants gained activity with NADP+ above the wild-type enzyme. And remarkably, it was found that all of the mutants investigated resulted in some degree of reversal of cofactor specificity in the oxidative direction. These changes were generally a result of changes in conformations of the ternary enzyme/cofactor/substrate complexes as opposed to changes in affinities or binding energies of the cofactors. This study highlights the role that amino acids which are distal to the cofactor binding pocket but are involved in substrate interactions can influence cofactor specificity in AdhD, and this strategy should translate to other AKR family members.

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