A Novel N-Acetylglutamate Synthase Architecture Revealed by the Crystal Structure of the Bifunctional Enzyme from Maricaulis maris

Abstract

Novel bifunctional N-acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N-acetylglutamate synthase (NAGS), an essential cofactor-producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. Arginine is an allosteric inhibitor of NAGS but not NAGK activity. The crystal structure of M. maris NAGS/K (mmNAGS/K) at 2.7 Å resolution indicates that it is a tetramer, in contrast to the hexameric structure of Neisseria gonorrhoeae NAGS. The quaternary structure of crystalline NAGS/K from Xanthomonas campestris (xcNAGS/K) is similar, and cross-linking experiments indicate that both mmNAGS/K and xcNAGS are tetramers in solution. Each subunit has an amino acid kinase (AAK) domain, which is likely responsible for N-acetylglutamate kinase (NAGK) activity and has a putative arginine binding site, and an N-acetyltransferase (NAT) domain that contains the putative NAGS active site. These structures and sequence comparisons suggest that the linker residue 291 may determine whether arginine acts as an allosteric inhibitor or activator in homologous enzymes in microorganisms and vertebrates. In addition, the angle of rotation between AAK and NAT domains varies among crystal forms and subunits within the tetramer. A rotation of 26° is sufficient to close the predicted AcCoA binding site, thus reducing enzymatic activity. Since mmNAGS/K has the highest degree of sequence homology to vertebrate NAGS of NAGS and NAGK enzymes whose structures have been determined, the mmNAGS/K structure was used to develop a structural model of human NAGS that is fully consistent with the functional effects of the 14 missense mutations that were identified in NAGS-deficient patients.

Effect of arginine on oligomerization and stability of N-acetylglutamate synthase

“…1). Because both MmNAGS-K and XcNAGS-K are completely inhibited by L-arginine1215 synthase activity of both enzymes was zero after incubation at temperatures between 20 °C and 70 °C in the presence of 1 mM L-arginine (Fig. 2).…”

“…Earlier studies of NAGS from bacteria, fish and mammals revealed that the oligomeric state of some of these proteins changes upon binding of L-arginine112532, while others do not13151624. Because the three-dimensional structure of NgNAGS, whose oligomerization state does not change upon binding of L-arginine, is known1316, while the structure of NAGS from E. coli , whose oligomerization state does change in the presence of L-arginine25 remains elusive, we tested if a similar correlation holds for vertebrate-like MmNAGS-K and XcNAGS-K, both with known three-dimensional structures, though the resolution of XcNAGS-M crystals is lower than the MmNAGS-K crystals15.…”

“…Phylogenetic analysis of NAGS from different organisms revealed two families of NAGS proteins: a classical, or E. coli -like, NAGS from most bacteria and plants, and a vertebrate-like NAGS from some bacteria, fungi and animals12. The NAGS polypeptide consists of two conserved structural domains, an amino acid kinase domain that harbors the binding site for the allosteric regulator L-arginine and an N-acetyltransferase domain that binds substrates and contains the catalytic site5121314151617.…”

“…However, the E. coli -like NAGS from Neisseria gonorrhoeae (NgNAGS) is a hexamer both in the absence and presence of L-arginine1316. The crystal structure of the vertebrate-like NAGS-K from Xanthomonas campestris (XcNAGS-K) revealed a tetrameric oligomerization state in the absence of L-arginine15; vertebrate-like NAGS-K from Maricaulis maris (MmNAGS-K) and Saccharomyces cerevisiae N-acetylglutamate kinase, which are highly similar to vertebrate NAGS12 are tetramers, both with, and without, L-arginine152426. Unlike its bacterial counterparts, purified rat NAGS appears to change oligomerization state in the presence of L-arginine9.…”

“…Although the three dimensional structures of two bacterial NAGS and a fungal NAGK have been determined152426, the structure of mammalian NAGS remains elusive, possibly because they exist as an ensemble of different oligomers in solution. Therefore, we compared the effect of L-arginine on the oligomerization state and stability of mouse NAGS and bacterial homologs with known three-dimensional structures.…”