Structure-Based Engineering of Amidase from Pantoea sp. for Efficient 2-Chloronicotinic Acid Biosynthesis

2-Chloronicotinic acid is a key intermediate of pharmaceuticals and pesticides. Amidase-catalyzed hydrolysis provides a promising enzymatic method for 2-chloronicotinic acid production from 2-chloronicotinamide. However, biocatalytic hydrolysis of 2-chloronicotinamide is difficult due to the strong steric and electronic effect caused by 2-position chlorine substituent of the pyridine ring. In this study, an amidase from a Pantoea sp. (Pa-Ami) was designed and engineered to have improved catalytic properties. Single mutant G175A and double mutant G175A/A305T strains exhibited 3.2- and 3.7-fold improvements in their specific activity for 2-chloronicotinamide, and the catalytic efficiency was significantly increased, with k_cat/K values 3.1 and 10.0 times higher than that of the wild type, respectively. Structure-function analysis revealed that the distance between Oγ of Ser177 (involved in the catalytic triad) and the carbonyl carbon of 2-chloronicotinamide was shortened in the G175A mutant, making the nucleophilic attack on the Oγ of Ser177 easier by virtue of proper orientation. In addition, the A305T mutation contributed to a suitable tunnel formation to facilitate the substrate entry and product release, resulting in improved catalytic efficiency. With the G175A/A305T double mutant as a biocatalyst, a maximum of 1,220 mM 2-chloronicotinic acid was produced with a 94% conversion, and the space-time yield reached as high as 575 g_product liter^-1 day^-1 These results provide not only a novel robust biocatalyst for the production of 2-chloronicotinic acid but also new insights into amidase structure-function relationships.IMPORTANCE In recent years, the demand for 2-chloronicotinic acid has been greatly increased. To date, several chemical methods have been used for the synthesis of 2-chloronicotinic acid, but all include tedious steps and/or drastic reaction conditions, resulting in both economic and environmental issues. It is requisite to develop an efficient and green synthesis route. We recently screened Pa-Ami and demonstrated its potential for synthesis of 2-chloronicotinic acid from 2-chloronicotinamide. However, chlorine substitution on the pyridine ring of nicotinamide significantly affected the activity of Pa-Ami. Especially for 2-chloronicotinamide, the enzyme activity and catalytic efficiency were relatively low. In this study, based on structure-function analysis, we succeeded in engineering the amidase by structure-guided saturation mutagenesis. The engineered Pa-Ami exhibited quite high catalytic activity toward 2-chloronicotinamide and could serve as a promising biocatalyst for the biosynthesis of 2-chloronicotinic acid.