L-amino acid ligases (Lals) are promising biocatalysts for the synthesis of dipeptides with special biological properties. However, their poor (or broad) substrate specificity limits their industrial applications. To address this problem, a molecular engineering method for Lals was developed to enhance their catalytic performance. Based on substrate channeling, entrances to the active site for different substrates were identified, and the "gate" located around the active site pocket, which plays an essential role in substrate recognition, was then engineered to facilitate acceptance of L-Gln. Two mutants (L110Y and N108F/L110Y) were discovered to display significantly increased catalytic activity toward L-Ala and L-Gln in the biosynthesis of Ala-Gln. The catalytic efficiency (kcat/ Km) of the L110Y and N108F/L110Y mutants was improved by 2.64-fold and 4.06-fold, respectively, compared with that of the wild type. N108F/L110Y was then further applied for batch production of Ala-Gln, which showed that the released Pi yield was 694.47 μM, which was an increase of approximately 21.4 %, and the yield of Ala-Gln was approximately 2.59 mM-1 L-1 mg-1. Collectively, these findings suggest the potential practical application of this method in the rational design of Lals for increased catalytic performance.
Keywords: (L)-amino acid ligase; Ala-Gln; Rational design; Substrate channeling; Substrate recognition.
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