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

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.