Structure-Based Engineering of an Artificially Generated NADP(+)-Dependent D-Amino Acid Dehydrogenase

摘要

A stable NADP(+)-dependent D-amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericus meso-diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 angstrom, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized D-phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 mu mol/min/mg at 50 degrees C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward D-leucine, D-norleucine, D-methionine, D-isoleucine, and D-tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 mu mol/min/mg at 50 degrees C). The structures of the D94A-Y224F double mutant in complex with NADP(+) and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 angstrom and 1.74 angstrom, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic D-amino acids with bulky side chains.