Directioned Evolution Of Microbial Arginine Deiminase By Site-directed Mutagenesis

Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In order to have an insight into the effect of the key mutation sites of M314, the molecular mechanism of arginine deiminase activity was studied by site-directed mutagenesis.Three mutation sites A128, H404 and I410 were introduced into wide-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L) and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R have a remarkable effect on the enhanced optimum pH, higher activity and stability of ADI at pH 7.4, as well as reduced Km value.In order to further improve the ADI properties, V10F, R18L, D38H, D44E, A128R and E296K were selected as potential mutation sites by sequence analysis and references, Six mutation sites V10F, R18L, D38H, D44E, A128R and E296K were introduced into wide-type ADI gene by site-directed mutagenesis method, and the enzymatic properties of mutant proteins were determined. The results show that both D38H and E296K have the remarkable effect on the higher activity of ADI under physiological condition. Both mutation sites, D38H and E296K were introduced into M314, and three mutants M11 (D38H/A128T/H404R/I410L), M12 (A128T/E296K/H404R/I410L) and M13 (D38H/A128T/E296K/H404R/I410L) were obtained. The specific activity of M13 (17.02 U/mg) was 58% higher than M314 (10.08 U/mg) at pH 7.4, and the optimum pH was 6.5. Km of M13 (0.57 mmol/L) was low than M314 (0.67 mmol/L) at pH 7.4. kcat/Km value of M13 was also significantly higher than M314. The results show that M13 have a remarkable effect on substrate affinity and catalytic activity of enzyme.Based on three-dimensinonal structure model of ADI from Pseudomonas aeruginosa, the homologous structure model of ADI mutant M314 was constructed with Swiss-Model. And three-dimensional structure of the mutation sites V10F, R18L, D38H, D44E, A128R, A128T, E296K, H404R and I410L were analyzed. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.