Site‐directed mutagenesis studies of acetylglutamate synthase delineate the site for the arginine inhibitor

Abstract

N-acetyl-L-glutamate synthase (NAGS), the first enzyme of bacterial/plant arginine biosynthesis and an essential activator of the urea cycle in animals, is, respectively, arginine-inhibited and activated. Site-directed mutagenesis of recombinant Pseudomonas aeruginosa NAGS (PaNAGS) delineates the arginine site in the PaNAGS acetylglutamate kinase-like domain, and, by extension, in human NAGS. Key residues for glutamate binding are identified in the acetyltransferase domain. However, the acetylglutamate kinase-like domain may modulate glutamate binding, since one mutation affecting this domain increases the K(m) for glutamate. The effects on PaNAGS of two mutations found in human NAGS deficiency support the similarity of bacterial and human NAGSs despite their low sequence identity.

Mechanism of Allosteric Inhibition of N-Acetyl-L-glutamate Synthase by L-Arginine

“…In addition, at both L-arginine concentrations, binding becomes cooperative, with a Hill coefficient of 1.5 Ϯ 0.2, providing evidence for an allosteric transition from an R-to T-state. These data agree well with earlier results for paNAGS and NAGS from other organisms (22)(23)(24)(25). They clearly indicate that L-arginine alters both the affinity of the enzyme for L-glutamate and the catalytic efficiency of the enzyme, and thus are consistent with the conclusions drawn from the crystal structures.…”

“…280 and Glu 334 , located around the side chain of L-arginine, may also be important for L-arginine binding. The location and orientation of the L-arginine binding in ngNAGS is the same as that identified in tmNAGK (5), and conservation of the L-arginine site between NAGS and NAGK is supported by sitedirected mutagenesis studies of recombinant paNAGS (25), mouse NAGS and bifunctional Xanthomonas campestris (xc) NAGS/K (27). Mutation of key residues binding L-arginine significantly decreased inhibition but had little effect on the kinetic parameters of the protein.…”

“…The L-arginine-binding sequence signature, E(I/L)(F/ M)(T/S)XXGXGTX, can be used not only to identify L-arginine-sensitive NAGK (5) but also to identify L-arginine-binding sites in NAGS proteins across phyla. Mutagenesis studies of this region in mouse NAGS and bifunctional xcNAGS/K (27) and paNAGS (25) support the universality of the L-arginine-binding site in NAGS and NAGK. The conservation of this site in mammalian NAGS is noteworthy, because L-arginine activates mammalian NAGS.…”

Functional Dissection of N -Acetylglutamate Synthase (ArgA) of Pseudomonas aeruginosa and Restoration of Its Ancestral N -Acetylglutamate Kinase Activity

“…Although the key role and widespread distribution of NAGS in all domains of life warrant studies of this enzyme, most detailed data concern the bacterial forms of this enzyme (17,23,27,32,33), including the crystal structure of NAGS from Neisseria gonorrhoeae (NgNAGS) in the substrate-bound and arginine-bound forms (24,34). NgNAGS can be considered a typical example of classical bacterial NAGSs, as defined by the early-studied Escherichia coli and Pseudomonas aeruginosa enzymes (17,23,32,33).…”

“…NgNAGS can be considered a typical example of classical bacterial NAGSs, as defined by the early-studied Escherichia coli and Pseudomonas aeruginosa enzymes (17,23,32,33). These classical bacterial forms, encoded by argA ( Fig.…”