Affiliation:
1. Instituto de Biomedicina de Valencia (IBV-CSIC)
2. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER-ISCIII), C/Jaime Roig 11, 46010 Valencia, Spain
Abstract
ABSTRACT
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 αH-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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
26 articles.
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