P79: Novel meso-2,6-D-Diaminopimelic Acid Dehydrogenase with expanded Substrate Specificity

D-Amino acids are important building blocks in the production of pharmaceuticals and fine chemicals. D-Amino acid dehydrogenases (D-AADH) catalyze the reductive amination of 2-keto acid with ammonia to form D-amino acid, offering a highly useful and complementary approach to the current methods. However, D-amino acid dehydrogenases are not ubiquitous in nature. meso-Diaminopimelate dehydrogenase (meso-DAPDH, EC1.4.1.16), which catalyzes the reversible oxidative deamination of meso-2,6-diaminopimelate (meso-DAP) to yield L-2-amino-6-oxopimelate, is the only known NAD(P)-dependent dehydrogenase able to stereoselectively act on the D-configuration amino group of meso-DAP. This family of enzymes have been reported to be absolutely specific to meso-DAP. Therefore, it is highly desirable to search for D-amino acid dehydrogenases active towards a broad range of substrates.  Recently, we have cloned and expressed a meso-diaminopimelate dehydrogenase from Symbiobacterium therophilum. In addition to the activity towards the suggested native substrate meso-DAP, this enzyme was found to catalyze the reductive amination of 2-keto acids and 2-keto esters to furnish D-amino acids with enantiomeric excess up to 99%. To our knowledge, it is the first meso-diaminopimelate dehydrogenase reported with a broad substrate profile. The S. therophilum meso-DAPDH was thus used as the starting point for rational mutagenesis to further expand its substrate spectrum. Mutations of several residues in the substrate binding site were performed and mutant enzymes with expanded substrate profile were obtained. These enzymes are potentially useful for the production of D-amino acids via the reductive amination of the corresponding 2-oxo acid with ammonia.