title: Site-directed mutagenesis studies on the L-arginine-binding sites of feedback inhibition in N-acetyl-L-glutamate kinase (NAGK) from Corynebacterium glutamicum.
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 I₀.₅ (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.
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province 214122, People's Republic of China.
authors.forename: Meijuan
authors.initials: M
authors.lastname: Xu
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Rao
authors.forename: Wenfang
authors.initials: W
authors.lastname: Dou
authors.forename: Jian
authors.initials: J
authors.lastname: Jin
authors.forename: Zhenghong
authors.initials: Z
authors.lastname: Xu
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D048230:Corynebacterium glutamicum / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D025461:Feedback, Physiological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D011485:Protein Binding; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D001120:Arginine; D017851:Phosphotransferases (Carboxyl Group Acceptor); C018721:acetylglutamate kinase
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