A Novel meso -Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum: Overexpression, Characterization, and Potential for d -Amino Acid Synthesis

Abstract

ABSTRACT meso -Diaminopimelate dehydrogenase ( meso -DAPDH) is an NADP + -dependent enzyme which catalyzes the reversible oxidative deamination on the d -configuration of meso -2,6-diaminopimelate to produce l -2-amino-6-oxopimelate. In this study, the gene encoding a meso -diaminopimelate dehydrogenase from Symbiobacterium thermophilum was cloned and expressed in Escherichia coli . In addition to the native substrate meso -2,6-diaminopimelate, the purified enzyme also showed activity toward d -alanine, d -valine, and d -lysine. This enzyme catalyzed the reductive amination of 2-keto acids such as pyruvic acid to generate d -amino acids in up to 99% conversion and 99% enantiomeric excess. Since meso -diaminopimelate dehydrogenases are known to be specific to meso -2,6-diaminopimelate, this is a unique wild-type meso -diaminopimelate dehydrogenase with a more relaxed substrate specificity and potential for d -amino acid synthesis. The enzyme is the most stable meso -diaminopimelate dehydrogenase reported to now. Two amino acid residues (F146 and M152) in the substrate binding sites of S. thermophilum meso -DAPDH different from the sequences of other known meso -DAPDHs were replaced with the conserved amino acids in other meso -DAPDHs, and assay of wild-type and mutant enzyme activities revealed that F146 and M152 are not critical in determining the enzyme's substrate specificity. The high thermostability and relaxed substrate profile of S. thermophilum meso -DAPDH warrant it as an excellent starting enzyme for creating effective d -amino acid dehydrogenases by protein engineering.