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

Biocatalysis - Original Paper
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Abstract

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 6.3.2.4) 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.

Keywords

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

Notes

Acknowledgments

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)

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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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