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Journal of Bacteriology, April 2008, p. 3018-3025, Vol. 190, No. 8
0021-9193/08/$08.00+0     doi:10.1128/JB.01831-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Basis of Arginine Sensitivity of Microbial N-Acetyl-L-Glutamate Kinases: Mutagenesis and Protein Engineering Study with the Pseudomonas aeruginosa and Escherichia coli Enzymes{triangledown}

M. Leonor Fernández-Murga1 and Vicente Rubio1,2*

Instituto de Biomedicina de Valencia (IBV-CSIC),1 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER-ISCIII), C/Jaime Roig 11, 46010 Valencia, Spain2

Received 20 November 2007/ Accepted 2 February 2008

N-Acetylglutamate kinase (NAGK) catalyzes the second step of arginine biosynthesis. In Pseudomonas aeruginosa, but not in Escherichia coli, this step is rate limiting and feedback and sigmoidally inhibited by arginine. Crystal structures revealed that arginine-insensitive E. coli NAGK (EcNAGK) is homodimeric, whereas arginine-inhibitable NAGKs, including P. aeruginosa NAGK (PaNAGK), are hexamers in which an extra N-terminal kinked helix (N-helix) interlinks three dimers. By introducing single amino acid replacements in PaNAGK, we prove the functionality of the structurally identified arginine site, as arginine site mutations selectively decreased the apparent affinity for arginine. N-helix mutations affecting R24 and E17 increased and decreased, respectively, the apparent affinity of PaNAGK for arginine, as predicted from enzyme structures that revealed the respective formation by these residues of bonds favoring inaccessible and accessible arginine site conformations. N-helix N-terminal deletions spanning ≥16 residues dissociated PaNAGK to active dimers, those of ≤20 residues decreased the apparent affinity for arginine, and complete N-helix deletion (26 residues) abolished arginine inhibition. Upon attachment of the PaNAGK N-terminal extension to the EcNAGK N terminus, EcNAGK remained dimeric and arginine insensitive. We concluded that the N-helix and its C-terminal portion after the kink are essential but not sufficient for hexamer formation and arginine inhibition, respectively; that the N-helix modulates NAGK affinity for arginine and mediates signal transmission between arginine sites, thus establishing sigmoidal arginine inhibition kinetics; that the mobile {alpha}H-β16 loop of the arginine site is the modulatory signal receiver; and that the hexameric architecture is not essential for arginine inhibition but is functionally essential for physiologically relevant arginine control of NAGK.

* Corresponding author. Mailing address: Instituto de Biomedicina de Valencia (IBV-CSIC), C/Jaime Roig 11, 46010 Valencia, Spain. Phone: 34 96 339 17 72. Fax: 34 96 369 08 00. E-mail: rubio{at}ibv.csic.es

{triangledown} Published ahead of print on 8 February 2008.

Journal of Bacteriology, April 2008, p. 3018-3025, Vol. 190, No. 8
0021-9193/08/$08.00+0     doi:10.1128/JB.01831-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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