Cells using lysis buffer. (DOC)Author ContributionsConceived and designed the experiments: CAG RD SQ SGG PF AM. Performed the experiments: CAG BV. Analyzed the data: CAG RD BV SQ PF AM. Contributed reagents/materials/analysis tools: SQ SGG PF AM. Wrote the paper: CAG PF AM. Revising the article critically for MedChemExpress K162 important intellectual content: SGG SQ PF AM. Final approval of the version to be published. CAG RD BV SQ SGG PF AM.
N-acetylglutamate synthase (NAGS, EC 2.3.1.1) catalyzes the conversion of AcCoA and glutamate to CoA and N-acetylglutamate (NAG). In microorganisms and 10457188 plants, NAG is further converted to NAG phosphate by NAG kinase (NAGK, EC 2.7.2.8) to continue the L-arginine biosynthetic pathway [1,2]. However, in mammals, NAG has an entirely different role as the essential cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle [3]. Perhaps because NAG plays different roles in lower organisms and mammals, L-arginine has opposing regulatory effects on their NAGS POR8 site enzymes. In bacteria, particularly those that use the linear pathway for L-arginine biosynthesis, NAGS is feedback inhibited by the end product, L-arginine. Conversely, in mammals, L-arginine enhances the NAGS activity [3]. Phylogenetic analysis of NAGS protein sequences classifies them into two distinct types: bacteria-like, classic NAGS and vertebratelike NAGS [4]. Most bacterial and plant NAGS belong to the former, with high sequence similarity to Escherichia coli NAGS. The second type includes not only vertebrate NAGS, but also fungal NAGS and NAGK, and bacterial bifunctional NAGS/K. Nevertheless, in spite of structural similarities of the second typeNAGS of various species, it is still inhibited by L-arginine in microorganisms that utilize it. Previously, we determined the structure of NAGS from N. gonorrhoeae (ngNAGS) and showed that this type of NAGS has a hexameric quaternary structure and that each subunit has two distinct domains: an N-terminal amino acid kinase (AAK) domain and a C-terminal N-acetyltransferase (NAT) domain [5]. The AAK domain has a structure similar to those of various N-acetylglutamate kinases (NAGK), but it is devoid of NAGK activity. It also has an L-arginine binding site similar to those in L-arginine sensitive NAGK structures [6]. The NAT domain has a typical GCN5-related NAT fold and a site that catalyzes NAG synthesis ?which is located .25 A away from the L-arginine binding site [7]. We have also previously determined the structures of bifunctional NAGS/K from Maricaulis maris (mmNAGS/K) and Xanthomonas campestris (xcNAGS/K) [8]. Surprisingly, bifunctional NAGS/K oligomerizes to form a novel tetramer. Although the subunits of NAGS/K have similar structures to ngNAGS subunits with two distinct domains, their domain-domain linkers and relative domain orientations are different from those of ngNAGS. Inhibition by L-arginine of NAGS/K was proposed to result fromStructure of Human N-Acetyl-L-Glutamate Synthasechanges in the relative orientations of AAK and NAT domains that close the AcCoA binding site. Even though extensive efforts have been made to determine the mammalian NAGS structure, it has proven challenging because the complete protein is unstable in solution. We succeeded in obtaining stable and functional human NAGS NAT domain ?(hNAT) (residues 377?34) with NAG bound at 2.1 A resolution. This structure and related mutagenesis experiments allowed us to define the catalytic mechanism. We have also confirmed by crosslinking.Cells using lysis buffer. (DOC)Author ContributionsConceived and designed the experiments: CAG RD SQ SGG PF AM. Performed the experiments: CAG BV. Analyzed the data: CAG RD BV SQ PF AM. Contributed reagents/materials/analysis tools: SQ SGG PF AM. Wrote the paper: CAG PF AM. Revising the article critically for important intellectual content: SGG SQ PF AM. Final approval of the version to be published. CAG RD BV SQ SGG PF AM.
N-acetylglutamate synthase (NAGS, EC 2.3.1.1) catalyzes the conversion of AcCoA and glutamate to CoA and N-acetylglutamate (NAG). In microorganisms and 10457188 plants, NAG is further converted to NAG phosphate by NAG kinase (NAGK, EC 2.7.2.8) to continue the L-arginine biosynthetic pathway [1,2]. However, in mammals, NAG has an entirely different role as the essential cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle [3]. Perhaps because NAG plays different roles in lower organisms and mammals, L-arginine has opposing regulatory effects on their NAGS enzymes. In bacteria, particularly those that use the linear pathway for L-arginine biosynthesis, NAGS is feedback inhibited by the end product, L-arginine. Conversely, in mammals, L-arginine enhances the NAGS activity [3]. Phylogenetic analysis of NAGS protein sequences classifies them into two distinct types: bacteria-like, classic NAGS and vertebratelike NAGS [4]. Most bacterial and plant NAGS belong to the former, with high sequence similarity to Escherichia coli NAGS. The second type includes not only vertebrate NAGS, but also fungal NAGS and NAGK, and bacterial bifunctional NAGS/K. Nevertheless, in spite of structural similarities of the second typeNAGS of various species, it is still inhibited by L-arginine in microorganisms that utilize it. Previously, we determined the structure of NAGS from N. gonorrhoeae (ngNAGS) and showed that this type of NAGS has a hexameric quaternary structure and that each subunit has two distinct domains: an N-terminal amino acid kinase (AAK) domain and a C-terminal N-acetyltransferase (NAT) domain [5]. The AAK domain has a structure similar to those of various N-acetylglutamate kinases (NAGK), but it is devoid of NAGK activity. It also has an L-arginine binding site similar to those in L-arginine sensitive NAGK structures [6]. The NAT domain has a typical GCN5-related NAT fold and a site that catalyzes NAG synthesis ?which is located .25 A away from the L-arginine binding site [7]. We have also previously determined the structures of bifunctional NAGS/K from Maricaulis maris (mmNAGS/K) and Xanthomonas campestris (xcNAGS/K) [8]. Surprisingly, bifunctional NAGS/K oligomerizes to form a novel tetramer. Although the subunits of NAGS/K have similar structures to ngNAGS subunits with two distinct domains, their domain-domain linkers and relative domain orientations are different from those of ngNAGS. Inhibition by L-arginine of NAGS/K was proposed to result fromStructure of Human N-Acetyl-L-Glutamate Synthasechanges in the relative orientations of AAK and NAT domains that close the AcCoA binding site. Even though extensive efforts have been made to determine the mammalian NAGS structure, it has proven challenging because the complete protein is unstable in solution. We succeeded in obtaining stable and functional human NAGS NAT domain ?(hNAT) (residues 377?34) with NAG bound at 2.1 A resolution. This structure and related mutagenesis experiments allowed us to define the catalytic mechanism. We have also confirmed by crosslinking.