Mechanism of arginine deiminase activity by site-directed mutagenesis.
- Author:
Lifeng LI
1
;
Ye NI
;
Zhihao SUN
Author Information
1. Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
- Publication Type:Journal Article
- MeSH:
Escherichia coli;
metabolism;
Hydrolases;
genetics;
metabolism;
Mutagenesis, Site-Directed
- From:
Chinese Journal of Biotechnology
2012;28(4):508-519
- CountryChina
- Language:Chinese
-
Abstract:
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 this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and 1410, were introduced into wild-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 had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced K(m) value. 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.
