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;

【DOI】

【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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    References

    [1] Martínez-Rodríguez S, Martinez-Gómez AI, Rodríguez-Vico F, et al. Natural occurrence and industrial applications of D-amino acids: an overview [J]. Chemistry & Biodivers, 2010, 7 (6): 1531–1548

    [2] Strömstedt AA, Pasupuleti M, Schmidtchen A, et al. Evaluation of strategies for improving proteolytic resistance of antimicrobial peptides by using variants of EFK17, an internal segment of LL-37 [J]. Antimicrobial Agents and Chemotherapy, 2009, 53 (2): 593–602

    [3] Kovacs M, Schally AV, Csernus B, et al. Luteinizing hormone-releasing hormone (LH-RH) antagonist Cetrorelix down-regulates the m RNA expression of pituitary receptors for LH-RH by counteracting the stimulatory effect of endogenous LH-RH [J]. Proceedings of the National Academy of Sciences of the United States of America, 2001, 98 (4): 1829–1834

    [4] Coward RM, Carson CC. Tadalafil in the treatment of erectile dysfunction [J]. Therapeutics and Clinical Risk Management, 2008, 4 (6): 1315–1329

    [5] Xia Q, Huang YH, Lin X, et al. Highly sensitive D-alanine electrochemical biosensor based on functionalized multi-walled carbon nanotubes and D-amino acid oxidase [J]. Biochemical Engineering Journal, 2016, 113: 1–6

    [6] Snell EE, Guirard BM. Some interrelationships of pyridoxine, alanine and glycine in their effect on certain lactic acid bacteria [J]. Proceedings of the National Academy of Sciences of the United States of America, 1943, 29 (2): 66–73

    [7] Deaton DN, Graham KP, Gross JW, et al. Thiol-based angiotensin-converting enzyme 2 inhibitors: P1′ modifications for the exploration of the S1′ subsite [J]. Bioorganic & Medicinal Chemistry Letters, 2008, 18 (5): 1681–1687

    [8] Behrends M, Wagner S, Kopka K, et al. New matrix metalloproteinase inhibitors based on γ-fluorinated α-aminocarboxylic andα-aminohydroxamic acids [J]. Bioorganic & Medicinal Chemistry, 2015, 23 (13): 3809–3818

    [9] Ley SV, Priour A. Total synthesis of the cyclic peptide argyrin B [J]. European Journal of Organic Chemistry, 2002, 2002 (23): 3995–4004

    [10] Shaginian A, Rosen MC, Binkowski BF, et al. Solid-phase synthesis of dihydrovirginiamycin S1, a streptogramin Bantibiotic [J]. Chemistry—A European Journal, 2004, 10 (17): 4334–4340

    [11] Fraser BH, Mulder RJ, Perlmutter P. The total synthesis of pamamycin-607. Part 2: synthesis of the C6-C18 domain [J]. Tetrahedron, 2006, 62 (12): 2857–2867

    [12] Farmer JJ, Attygalle AB, Smedley SR, et al. Absolute configuration of insect-produced epilachnene [J]. Tetrahedron Letters, 1997, 38 (16): 2787–2790

    [13] Attygalle AB, SvatošA, Veith M, et al. Biosynthesis of epilachnene, a macrocyclic defensive alkaloid of the Mexican bean beetle [J]. Tetrahedron, 1999, 55 (4): 955–966

    [14] Lennox JR, Turner SC, Rapoport H. Enantiospecific synthesis of annulated nicotine analogues from D-glutamic acid. 7-Azabicyclo[2.2.1]heptano[2.3-c]pyridines [J]. The Journal of Organic Chemistry, 2001, 66 (21): 7078–7083

    [15] Ksander GM, Yuan AM, Diefenbacher CG, et al. Angiotensin converting enzyme inhibitors: N-substituted D-glutamic acid gamma-dipeptides [J]. Journal of Medicinal Chemistry, 1985, 28 (11): 1606–1611

    [16] de Dios A, Prieto L, Martín JA, et al. 4-Substituted D-glutamic acid analogues: the first potent inhibitors of glutamate racemase (Mur I) enzyme with antibacterial activity [J]. Journal of Medicinal Chemistry, 2002, 45 (20): 4559–4570

    [17] Koeller KM, Wong CH. Enzymes for chemical synthesis [J]. Nature, 2001, 409 (6817): 232–240

    [18] Hollmann F, Arends IWCE, Holtmann D. Enzymatic reductions for the chemist [J]. Green Chemistry, 2011, 13 (9): 2285–2314

    [19] Bezsudnova EY, Popov VO, Boyko KM. Structural insight into the substrate specificity of PLP fold type IVtransaminases [J]. Applied Microbiology and Biotechnology, 2020, 104 (6): 2343–2357

    [20] Aganyants H, Weigel P, Hovhannisyan Y, et al. Rational engineering of the substrate specificity of a thermostable D-hydantoinase (Dihydropyrimidinase) [J]. High-Throughput, 2020, 9 (1): 5

    [21] Wang RY, Li JF, Dang DY, et al. Bacterial production of maize and human serine racemases as partially active inclusion bodies for D-serine synthesis [J]. Enzyme and Microbial Technology, 2020, 137: 109547

    [22] Martínez-Rodríguez S, Soriano-Maldonado P, Gavira JA. N-succinylamino acid racemases: enzymatic properties and biotechnological applications [J]. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 2020, 1868 (4): 140377

    [23] Parmeggiani F, Casamajo AR, Colombo D, et al. Biocatalytic retrosynthesis approaches to D-(2,4,5-trifluorophenyl)alanine, key precursor of the antidiabetic sitagliptin [J]. Green Chemistry, 2019, 21 (16): 4368–4379

    [24] Abbott SD, Lane-Bell P, Sidhu KPS. Synthesis and testing of heterocyclic analogs of diaminopimelic acid (DAP) as inhibitors of DAP dehydrogenase and DAP epimerase [J]. Journal of the American Chemical Society, 1994, 116 (15): 6513–6520

    [25] Olsiewski PJ, Kaczorowski GJ, Walsh C. Purification and properties of D-amino acid dehydrogenase, an inducible membrane-bound iron-sulfur flavoenzyme from Escherichia coli B [J]. The Journal of Biological Chemistry, 1980, 255 (10): 4487–4494

    [26] Jones H, Venables WA. Solubilisation of D-amino acid dehydrogenase of Escherichia coli K12 and ist re-binding to envelope preparations [J]. Biochimie, 1983, 65 (3): 177–183

    [27] Tanigawa M, Shinohara T, Saito M, et al. D-amino acid dehydrogenase from Helicobacter pylori NCTC 11637 [J]. Amino Acids, 2010, 38 (1): 247–255

    [28] Vedha-Peters K, Gunawardana M, Rozzell JD, et al. Creation of a broad-range and highly stereoselective D-amino acid dehydrogenase for the one-step synthesis of D-amino acids [J]. Journal of the American Chemical Society, 2006, 128 (33): 10923–10929

    [29] Akita H, Doi K, Kawarabayasi Y, et al. Creation of a thermostable NADP+-dependent D-amino acid dehydrogenase from Ureibacillus thermosphaericus strain A1 meso-diaminopimelate dehydrogenase by site-directed mutagenesis [J]. Biotechnology Letters, 2012, 34 (9): 1693–1699

    [30] Gao XZ, Chen X, Liu WD, et al. A novel meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum: overexpression, characterization, and potential for D-amino acid synthesis [J]. Applied and Environmental Microbiology, 2012, 78 (24): 8595–8600

    [31] Zhao LM, Liu WD, Chen X, et al. Effect of residue Y76 on co-enzyme specificity of meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum [J]. Chinese Journal of Biotechnology, 2015, 31 (7): 1108–1118 (in Chinese)

    [32] Liu WD, Li Z, Huang CX, et al. Structural and mutational studies on the unusual substrate specificity of meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum [J]. Chem Bio Chem, 2014, 15 (2): 217–222

    [33] Cheng XK, Chen X, Feng JH, et al. Structure-guided engineering of meso-diaminopimelate dehydrogenase for enantioselective reductive amination of sterically bulky 2-keto acids [J]. Catalysis Science & Technology, 2018, 8 (19): 4994–5002

    [34] Gao XZ, Huang F, Feng JH, et al. Engineering the meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum by site saturation mutagenesis for D-phenylalanine synthesis [J]. Applied and Environmental Microbiology, 2013, 79 (16): 5078–5081

    [35] Mayer KM, Arnold FH. A colorimetric assay to quantify dehydrogenase activity in crude cell lysates [J]. Journal of Biomolecular Screening, 2002, 7 (2): 135–140

    [36] Hayashi J, Seto T, Akita H, et al. Structure-based engineering of an artificially generated NADP+-dependent D-amino acid dehydrogenase [J]. Applied and Environmental Microbiology, 2017, 83 (11): e00491–17

    [37] Akita H, Hayashi J, Sakuraba H, et al. Artificial thermostable D-amino acid dehydrogenase: creation and application [J]. Frontiers in Microbiology, 2018, 9: 1760

This Article

ISSN:0253-2654

CN: 11-1996/Q

Vol 47, No. 07, Pages 2119-2127

July 2020

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Abstract

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