title: A novel meso-Diaminopimelate dehydrogenase from Symbiobacterium thermophilum: overexpression, characterization, and potential for D-amino acid synthesis.
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.
authors.affiliation: National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
authors.forename: Xiuzhen
authors.initials: X
authors.lastname: Gao
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.forename: Weidong
authors.initials: W
authors.lastname: Liu
authors.forename: Jinhui
authors.initials: J
authors.lastname: Feng
authors.forename: Qiaqing
authors.initials: Q
authors.lastname: Wu
authors.forename: Ling
authors.initials: L
authors.lastname: Hua
authors.forename: Dunming
authors.initials: D
authors.lastname: Zhu
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000096:biosynthesis*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000596:Amino Acids; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; C020032:diaminopimelate dehydrogenase