Mutation of a key amino acid residue of meso-diaminopimelate dehydrogenase enhances the catalytic activity toward alkyl substituted 2-keto acids

CHENG Xin-Kuan1,2 CHEN Xi2 FENG Jin-Hui2 WU Qia-Qing2 ZHU Dun-Ming2

(1.Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education; Tianjin Key Laboratory of Industrial Microbiology; College of Biotechnology, Tianjin University of Science & Technology, Tianjin, China 300457)
(2.Tianjin Biocatalysis Technology Engineering Center; National Engineering Laboratory for Industrial Enzymes; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China 300308)

【Abstract】[Background] Efficient biosynthesis of D-amino acids is highly desired. Meso-diaminopimelate dehydrogenase (DAPDH) synthesizes D-amino acids from 2-keto acids and ammonia. [Objective] To increase the catalytic activity against the alkyl substituted 2-keto acids. [Methods] Based on structural analysis and mutation results from previously selected sites, the saturation mutagenesis was carried out at the amino acid residue H227 of DAPDH from Symbiobacterium thermophilum (StDAPDH). The resulting mutant library was subjected to screening using D-alanine, D-2-aminobutyric acid, D-norvaline, and D-glutamic acid as substrates. [Results] The mutants H227Q and H227N were obtained. Mutant H227Q was found to have 10.9-, 11.5-, 8.6- and 7.6-folds improved enzyme activity toward pyruvic acid, 2-oxobutyric acid, 2-oxovaleric acid and 2-ketoglutaric acid, respectively, compared to that of wild-type enzyme. The kinetic parameters indicated that mutant H227Q increased the turnover number of the enzyme and the affinity of the enzyme for the substrate simultaneously, so that the catalytic efficiency (kcat/Km) of pyruvic acid was 9.4 folds higher than that of wild-type enzyme. Molecular modeling analysis of interaction between mutant H227Q and product amino acid, indicates that glutamine at position H227 forms a hydrogen bond with the carboxylic acid of the amino acid, so that the distance between the α-hydrogen atom of product amino acid and C4 of coenzyme nicotinamide ring was shortened. [Conclusion] Directed evolution technology has been successfully used to improve the catalytic activity of DADPH for alkyl-substituted 2-keto acids, which is helpful for the development of new high-efficiency biocatalysts. These efforts also provide guidance for our future engineering of this enzyme about more challenging D-amino acids.

【Keywords】 Alkyl substituted 2-keto acid; Meso-diaminopimelate dehydrogenase; Saturation mutation; Molecular docking; D-amino acids;


【Funds】 National Natural Science Foundation of China (21778072) Tianjin Science and Technology Commission Project (15PTCYSY00020; 15PTGCCX00060) Tianjin Education Commission Scientific Research Project (2019KJ239)

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This Article


CN: 11-1996/Q

Vol 47, No. 07, Pages 2119-2127

July 2020


Article Outline



  • 1 Materials and methods
  • 2 Results and analysis
  • 3 Discussion and conclusion
  • References