Engineering an ATP-dependent d-Ala:d-Ala ligase for synthesizing amino acid amides from amino acids

Biocatalysis - Original Paper


We successfully engineered a new enzyme that catalyzes the formation of d-Ala amide (d-AlaNH2) from d-Ala by modifying ATP-dependent d-Ala:d-Ala ligase (EC from Thermus thermophilus, which catalyzes the formation of d-Ala-d-Ala from two molecules of d-Ala. The new enzyme was created by the replacement of the Ser293 residue with acidic amino acids, as it was speculated to bind to the second d-Ala of d-Ala-d-Ala. In addition, a replacement of the position with Glu performed better than that with Asp with regards to specificity for d-AlaNH2 production. The S293E variant, which was selected as the best enzyme for d-AlaNH2 production, exhibited an optimal activity at pH 9.0 and 40 °C for d-AlaNH2 production. The apparent K m values of this variant for d-Ala and NH3 were 7.35 mM and 1.58 M, respectively. The S293E variant could catalyze the synthesis of 9.3 and 35.7 mM of d-AlaNH2 from 10 and 50 mM d-Ala and 3 M NH4Cl with conversion yields of 93 and 71.4 %, respectively. This is the first report showing the enzymatic formation of amino acid amides from amino acids.


d-Ala:d-Ala ligase Amino acid amide Amino acid Protein engineering Homology modeling Docking simulation 



We thank Dr. Kimiyasu Isobe for his fruitful discussion, critical preparation and reading of the manuscript. S.O. acknowledges Prof. Hiroaki Tokiwa of Rikkyo University for computational and analytic support. This work was supported by the Exploratory Research for Advanced Technology (ERATO) Asano Active Enzyme Molecule Project of Japan Science and Technology Agency (JST).

Supplementary material

10295_2016_1833_MOESM1_ESM.docx (396 kb)
Fig. S1 The structures of the overall folding (A) and the substrate recognition sites (B) of TtDdL. The original PDB file is 2ZDQ [15]. The graphic was visualized with the Pymol program (DOCX 395 kb)
10295_2016_1833_MOESM2_ESM.docx (344 kb)
Fig. S2 SDS-PAGE of the E. coli soluble fractions containing recombinant TtDdL from S293X. Wt is wild type enzyme, and alphabets are amino acid codes of X in S293X variants (DOCX 343 kb)
10295_2016_1833_MOESM3_ESM.docx (128 kb)
Fig. S3. A typical LC-MS chromatogram and the MS/MS patterns of the reaction products from d-Ala. The S293D variant was incubated with 10 mM d-Ala, 0.1 M NH4Cl, 10 mM ATP, 5 mM MgCl2, and 0.1 M KCl at pH 9.0 and 40°C for 12hours. The reaction products were analyzed by LC-MS and MS/MS as described in Materials and methods. The peak corresponding to d-Ala-d-Ala was eluted at 15.8 minutes. The new peak detected at 11.4 minutes was identified as d-AlaNH2 by the retention time and the MS/MS pattern of the authentic compound. The peak at 12.4 minutes was identified as d-Ala-d-AlaNH2 by comparison of the MS/MS pattern (DOCX 127 kb)
10295_2016_1833_MOESM4_ESM.docx (44 kb)
Fig. S4. Effects of ATP concentration on the production of d-AlaNH2 by the S293E variant. The d-AlaNH2 production was performed by incubation at 40°C for 12 hours at pH 9.0 using 25 mM or 50 mM d-Ala and 3 M NH4Cl as substrate. ATP added into the reaction mixture was changed as follows: 25 mM or 50 mM ATP was added to 25 mM d-Ala, and 50 mM or 100 mM ATP was added to 50 mM d-Ala. The concentrations of d-AlaNH2, d-Ala-d-Ala and remaining d-Ala are shown as the black, gray and white bars, respectively. Means and 95% confidence limits are shown (DOCX 43 kb)
10295_2016_1833_MOESM5_ESM.docx (491 kb)
Fig. S5. Comparison of the d-Ala2 binding sites among crystal structure of EcDdL (coral; PDB ID 1IOW) and homology modeled TtDdL structures of wild type (gray), S293D variant (gold) and S293E variant (ice blue). The figure was prepared by superposition of EcDdL, homology modelled TtDdL (wild type, S293D and S293E variants) on TtDdL (PDB ID 2ZDQ). Broken red lines represent hydrogen bonds. The figure was produced using CCP4mg [17] (DOCX 490 kb)
10295_2016_1833_MOESM6_ESM.docx (255 kb)
Fig. S6. Comparison of the modelled S293 variants in closed form. S293D variant (gold) and S293E variant (ice blue). Broken red lines represent hydrogen bonds. The figure was produced using CCP4mg [17] (DOCX 254 kb)
10295_2016_1833_MOESM7_ESM.docx (336 kb)
Fig. S7. Comparison of the modelled S293 variants in closed form. S293I variant (cyan), S293M variant (pink) and S293V variant (purple). Broken lines represent distances. The figure was produced using CCP4mg [17] (DOCX 335 kb)
10295_2016_1833_MOESM8_ESM.docx (18 kb)
Supplementary material 8 (DOCX 17 kb)
10295_2016_1833_MOESM9_ESM.docx (18 kb)
Supplementary material 9 (DOCX 17 kb)
10295_2016_1833_MOESM10_ESM.docx (17 kb)
Supplementary material 10 (DOCX 16 kb)
10295_2016_1833_MOESM11_ESM.docx (17 kb)
Supplementary material 11 (DOCX 17 kb)


  1. 1.
    Asano Y (2010) Tools for enzyme discovery-industrial enzymes, biocatalysis and enzyme evolution. Man Ind Microbiol Biotechnol, 441–452Google Scholar
  2. 2.
    Asano Y, Yamaguchi S (2005) Dynamic kinetic resolution of amino acid amide catalyzed by D-aminopeptidase and α-amino-ε-caprolactam racemase. J Am Chem Soc 127:7696–7697CrossRefPubMedGoogle Scholar
  3. 3.
    Brennan TM, Hendrick ME (1983) Branched amides of l-aspartyl-d-amino acid dipeptides. US Patent 4:411–925Google Scholar
  4. 4.
    Breuer M, Ditrich K, Habicher T, Hauer B, Kesseler M, Stürmer R, Zelinski T (2004) Industrial methods for the production of optically active intermediates. Angew Chem Int Ed Engl 43:788–824CrossRefPubMedGoogle Scholar
  5. 5.
    Dutta AS (2009) Discovery of New Medicine. The Textbook of Pharmaceutical Medicine. (Griffin, JP. ed. 6th Edition). Wiley, Chichester, pp 3–80Google Scholar
  6. 6.
    Ghislieri D, Houghton D, Green AP, Willies SC, Turner NJ (2013) Monoamine oxidase (MAO-N) catalyzed deracemization of tetrahydro-β-carbolines: substrate dependent switch in enantioselectivity. ACS Catal 3:2869–2872CrossRefGoogle Scholar
  7. 7.
    Goto J, Kataoka R (2008) ASEDock-docking based on alpha spheres and excluded volumes. J Chem Inf Model 48:583–590CrossRefPubMedGoogle Scholar
  8. 8.
    Fan C, Moews PC, Walsh CT, Knox JR (1994) Vancomycin resistance: structure of d-alanine:d-alanine ligase at 2.3 Å resolution. Science 266:439–443CrossRefPubMedGoogle Scholar
  9. 9.
    Fan C, Park IS, Walsh CT, Knox JR (1997) d-Alanine:d-alanine ligase: phosphonate and phosphinate intermediates with wild type and the Y216F mutant. Biochemistry 36:2531–2538CrossRefPubMedGoogle Scholar
  10. 10.
    Goswami A, Van Lanen SG (2015) Enzymatic strategies and biocatalysts for amide bond formation: tricks of the trade outside of the ribosome. Mol BioSyst 11:338–353CrossRefPubMedGoogle Scholar
  11. 11.
    Gröger H, Asano Y (2012) Introduction-principles and historical landmarks of enzyme catalysis in organic synthesis. Enzyme Catalysis in Organic Synthesis, Third Edition, pp 1–42Google Scholar
  12. 12.
    Gröger H, Asano Y, Bornscheuer UT, Ogawa J (2012) Development of biocatalytic processes in Japan and Germany: from research synergies to industrial applications. Chem Asian J 7:1138–1153CrossRefPubMedGoogle Scholar
  13. 13.
    Kato Y, Asano Y, Nakazawa A, Kondo K (1989) First stereoselective synthesis of d-amino acid N-alkyl amide catalyzed by d-aminopeptidase. Tetrahedron 45:5743–5754CrossRefGoogle Scholar
  14. 14.
    Kawahara N, Asano Y (2015) Mutagenesis of an Asn156 residue in a surface region of S-selective hydroxynitrile lyase from Baliospermum montanum enhances catalytic efficiency and enantioselectivity. ChemBioChem 16:1891–1895CrossRefGoogle Scholar
  15. 15.
    Kitamura Y, Ebihara A, Agari Y, Shinkai A, Hirotsu K, Kuramitsu S (2009) Structure of d-alanine-D-alanine ligase from Thermus thermophilus HB8: cumulative conformational change and enzyme–ligand interactions. Acta Crystallogr D Biol Crystallogr 65:1098–1106CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Komeda H, Asano Y (1999) Synthesis of d-phenylalanine oligopeptides catalyzed by alkaline D-peptidase from Bacillus cereus DF4-B. J Mol Catal B Enzym 6:379–386CrossRefGoogle Scholar
  17. 17.
    McNicholas S, Potterton E, Wilson KS, Noble MEM (2011) Presenting your structures: the CCP4 mg molecular-graphics software. Acta Cryst. D67:386–394Google Scholar
  18. 18.
    Mullins L, Zawadzke L, Walsh CT, Raushel F (1990) Kinetic evidence for the formation of d-alanyl phosphate in the mechanism of d-alanyl-d-alanine ligase. J Biol Chem 265:8993–8998PubMedGoogle Scholar
  19. 19.
    Neuhaus FC (1962) The enzymatic synyhesis of D-alanyl-d-alanine: I. Purification and properties of D-alanyl-d-alanine synthetase. J Biol Chem 237:778–786PubMedGoogle Scholar
  20. 20.
    Sharma J, Batovska D, Kuwamori Y, Asano Y (2005) Enzymatic chemoselective synthesis of secondary-amide surfactant from N-methylethanol amine. J Biosci Bioeng 100:662–666CrossRefPubMedGoogle Scholar
  21. 21.
    Tsuda T, Asami M, Koguchi Y, Kojima S (2014) Single mutation alters the substrate specificity of l-amino acid ligase. Biochemistry 53:2650–2660CrossRefPubMedGoogle Scholar
  22. 22.
    van Rantwijk F, Hacking MA, Sheldon RA (2000) Lipase-catalyzed synthesis of carboxylic amides: nitrogen nucleophiles as acyl acceptor. Monatsh Chem 131:549–569CrossRefGoogle Scholar
  23. 23.
    Wehofsky N, Pech A, Liebscher S, Schmidt S, Komeda H, Asano Y, Bordusa F (2008) d-Amino acid specific proteases and native all-l-proteins: a convenient combination for semisynthesis. Angew Chem Int Ed Engl 47:5456–5460CrossRefPubMedGoogle Scholar
  24. 24.
    Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AGW, McCoy A, McNicholas SJ, Murshudov GN, Pannu NS, Potterton EA, Powell HR, Read RJ, Vagin A, Wilson S (2011) Overview of the CCP4 suite and current developmentts. Acta Cryst. D67:235–242Google Scholar
  25. 25.
    Yamaguchi S, Komeda H, Asano Y (2007) New enzymatic method of chiral amino acid synthesis by dynamic kinetic resolution of amino acid amides: use of stereoselective amino acid amidases in the presence of α-amino-ε-caprolactam racemase. Appl Environ Microbiol 73:5370–5373CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Yasukawa K, Asano Y (2012) Enzymatic synthesis of chiral phenylalanine derivatives by a dynamic kinetic resolution of corresponding amide and nitrile substrates with a multi-enzyme system. Adv Synth Catal 354:3327–3332CrossRefGoogle Scholar
  27. 27.
    Yasukawa K, Nakano S, Asano Y (2014) Tailoring d-amino acid oxidase from the pig kidney to R-stereoselective amine oxidase and its use in the deracemization of α-methylbenzylamine. Angew Chem Int Ed Engl 53:4428–4431CrossRefPubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2016

Authors and Affiliations

  1. 1.Biotechnology Research Center and Department of BiotechnologyToyama Prefectural UniversityImizuJapan
  2. 2.Asano Active Enzyme Molecule ProjectERATO, JSTImizuJapan
  3. 3.MicroBiopharm Japan Co.Ltd.TokyoJapan

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