Site-Directed Mutagenesis Studies on the l-Arginine-Binding Sites of Feedback Inhibition in N-Acetyl-l-glutamate Kinase (NAGK) from Corynebacterium glutamicum
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Arginine biosynthesis in Corynebacterium glutamicum proceeds via a pathway that is controlled by arginine through feedback inhibition of NAGK, the enzyme that converts N-acetyl-l-glutamate (NAG) to N-acety-l-glutamy-l-phosphate. In this study, the gene argB encoding NAGK from C. glutamicum ATCC 13032 was site-directed, and the l-arginine-binding sites of feedback inhibition in Cglu_NAGK are described. The N-helix and C-terminal residues were first deleted, and the results indicated that they are both necessary for Cglu_NAGK, whereas, the complete N-helix deletion (the front 28 residues) abolished the l-arginine inhibition. Further, we study here the impact on these functions of 12 site-directed mutations affecting seven residues of Cglu_NAGK, chosen on the basis of homology structural alignment. The E19R, H26E, and H268N variants could increase the I0.5 R 50–60 fold, and the G287D and R209A mutants could increase the I0.5 R 30–40 fold. The E281A mutagenesis resulted in the substrate kinetics being greatly influenced. The W23A variant had a lower specific enzyme activity. These results explained that the five amino acid residues (E19, H26, R209, H268, and G287) located in or near N-helix are all essential for the formation of arginine inhibition.
KeywordsFeedback Inhibition Corynebacterium Glutamicum Specific Enzyme Activity Arginine Biosynthesis Corynebacterium Diphtheriae
This study was supported by the High-tech Research and Development Programs of China (2007AA02Z207), the National Basic Research Program of China (2007CB707804), the National Natural Science Foundation of China (30970056), the Program for New Century Excellent Talents in University (NCET-07-0380, NCET-10-0459), the Fundamental Research Funds for the Central Universities (JUSRP31001), the Program of Introducing Talents of Discipline to Universities (111-2-06), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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