Current Microbiology

, Volume 64, Issue 2, pp 164–172 | Cite as

Site-Directed Mutagenesis Studies on the l-Arginine-Binding Sites of Feedback Inhibition in N-Acetyl-l-glutamate Kinase (NAGK) from Corynebacterium glutamicum

Article

Abstract

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 I0.5R 50–60 fold, and the G287D and R209A mutants could increase the I0.5R 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.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiPeople’s Republic of China
  2. 2.The Laboratory of Pharmaceutical Engineering, School of Medicine and PharmaceuticsJiangnan UniversityWuxiPeople’s Republic of China

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