Engineering an ATP-dependent d-Ala:d-Ala ligase for synthesizing amino acid amides from amino acids
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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 126.96.36.199) 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.
Keywordsd-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).
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