Abstract
N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in l-arginine biosynthesis in plants and micro-organisms and is subject to feedback inhibition by l-arginine. This study compares the crystal structures of NAGS from Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with l-arginine bound and in the active R-state complexed with CoA and l-glutamate. Under all of the conditions examined, the enzyme consists of two stacked trimers. Each monomer has two domains: an amino acid kinase (AAK) domain with an AAK-like fold but lacking kinase activity and an N-acetyltransferase (NAT) domain homologous to other GCN5-related transferases. Binding of l-arginine to the AAK domain induces a global conformational change that increases the diameter of the hexamer by approximately 10 A and decreases its height by approximately 20A(.) AAK dimers move 5A outward along their 2-fold axes, and their tilt relative to the plane of the hexamer decreases by approximately 4 degrees . The NAT domains rotate approximately 109 degrees relative to AAK domains enabling new interdomain interactions. Interactions between AAK and NAT domains on different subunits also change. Local motions of several loops at the l-arginine-binding site enable the protein to close around the bound ligand, whereas several loops at the NAT active site become disordered, markedly reducing enzymatic specific activity.
Highlights
L-Arginine biosynthesis in most micro-organisms and plants involves the initial acetylation of L-glutamate by N-acetylglutamate synthase (NAGS, EC 2.3.1.1)2 to produce N-acetylgluta
L-Arginine-insensitive Escherichia coli NAGK is a homodimer [4], whereas L-arginine-sensitive NAGKs from Thermotoga maritima and Pseudomonas aeruginosa are hexamers formed by pair-wise interlacing of the N-terminal helices of three ecNAGK-like dimers, to create a second type of dimer interface
L-Arginine binding to a site close to the C terminus induces global conformational changes that expands the ring by ϳ8 Å and decreases the tilt of the ecNAGK-like dimers relative to the plane of the ring by ϳ6°
Summary
Enzyme Production and Crystallization—Cloning, expression, purification, and crystallization of ngNAGS have been previously described [6]. In the absence of L-arginine, AcCoA or L-glutamate concentration was varied in the range of 0.05– 0.5 and 0.5– 6.0 mM, respectively, with L-glutamate and AcCoA concentrations fixed at 15 or 4 mM, respectively (see Fig. 1, A and B) These titration data were fit to MichaelisMenten kinetics using the program GNUPLOT. The L-glutamate titration data measured in the presence of 0.1 and 1.0 mM of L-arginine were fit to sigmoidal kinetics, using the equation: v ϭ Vmax ϫ [Glu]n/(Kmn ϩ [Glu]n), where v and Vmax are activities at a given [Glu] and at [Glu] ϭ ∞; Km is half-maximal activity [Glu]; and n is the Hill coefficient. In the presence of 1.0 mM of L-arginine, L-glutamate concentrations were varied in the range 0.5–140.0 mM, and 1.6 g of enzyme was used At both L-arginine concentrations, AcCoA was fixed at 4 mM (see Fig. 1D). Resolution range (Å) No of protein atoms No of water atoms No of hetero atoms rmsd of bond lengths (Å) rmsd of bond angle (°) Rwork (%)b Rfree (%)c Ramachandran plot (%)
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