A Novelmeso-Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum: Overexpression, Characterization, and Potential for d-Amino Acid Synthesis
ABSTRACTmeso-Diaminopimelate dehydrogenase (meso-DAPDH) is an NADP+-dependent enzyme which catalyzes the reversible oxidative deamination on thed-configuration ofmeso-2,6-diaminopimelate to producel-2-amino-6-oxopimelate. In this study, the gene encoding ameso-diaminopimelate dehydrogenase fromSymbiobacterium thermophilumwas cloned and expressed inEscherichia coli. In addition to the native substratemeso-2,6-diaminopimelate, the purified enzyme also showed activity towardd-alanine,d-valine, andd-lysine. This enzyme catalyzed the reductive amination of 2-keto acids such as pyruvic acid to generated-amino acids in up to 99% conversion and 99% enantiomeric excess. Sincemeso-diaminopimelate dehydrogenases are known to be specific tomeso-2,6-diaminopimelate, this is a unique wild-typemeso-diaminopimelate dehydrogenase with a more relaxed substrate specificity and potential ford-amino acid synthesis. The enzyme is the most stablemeso-diaminopimelate dehydrogenase reported to now. Two amino acid residues (F146 and M152) in the substrate binding sites ofS. thermophilum meso-DAPDH different from the sequences of other knownmeso-DAPDHs were replaced with the conserved amino acids in othermeso-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 ofS. thermophilum meso-DAPDH warrant it as an excellent starting enzyme for creating effectived-amino acid dehydrogenases by protein engineering.