N-Acetyl-d-Glucosamine-6-Phosphate Deacetylase: Substrate Activation via a Single Divalent Metal Ion

NagA1 catalyzes the hydrolytic cleavage of N-acetyl-d-glucosamine-6-phosphate as illustrated in Scheme 1. The deacetylation of this compound provides a source of carbon and nitrogen by preparing this substrate for entry into the glycolytic pathway. This reaction is a key step in the catabolism of N-acetyl-d-glucosamine, derived from the degradation of chitin, and is an essential component for the biosynthesis of lipopolysaccharides and peptidoglycans. More recently, the reaction catalyzed by NagA has been shown to be an important step in the recycling of cell wall murein (1-3). Scheme 1 The purification of NagA from Escherichia coli was originally reported by White and Pasternak (4). The enzyme oligomerizes as a tetramer and each subunit contains a reactive sulfhydryl group near the active site (5). In the forward reaction, inhibition occurs at high substrate concentrations while the products, d-glucosamine-6-phosphate and acetate, are noncompetitive and competitive inhibitors, respectively. A phosphonate analogue of the putative tetrahedral intermediate has been synthesized and it is a potent, tight-binding inhibitor (6). The structure of apo-NagA from E. coli was first reported by the team led by Steve Almo (pdb code: 1ymy) and later by Ferreira et al. (7) (pdb code: 1yrr). The protein folds as a (β/α)8-barrel and is a member of the amidohydrolase superfamily (8, 9). The enzyme binds up to 1.4 equivalents of zinc and the metal can be removed with a chelating agent, resulting in the loss of activity that can be restored after reconstitution with Zn, Co, Mn or Fe (7). The precise role of the metal cofactor is of particular interest for NagA because the two crystal structures available for the enzyme from E. coli have no metal bound in the active site. However, the crystal structures of NagA from B. subtilis and T. maritima have been determined with divalent cations bound within the active site (10). The B. subtilis enzyme (pdb code: 1un7) binds two equivalents of Fe2+ (10). One of the metals is coordinated to the H×H motif at the end of β-strand 1 and an aspartic acid from the end of β-strand 8. This site has been designated as the Mα-position (8). The second metal (Mβ-site) is coordinated to two histidine residues from the ends of β-strands 5 and 6, respectively. The two metal ions are bridged to one another by a glutamate from the end of β-strand 3 and a hydroxide (or water) from solvent. The active site of the T. maritima enzyme is quite similar to that of the B. subtilis structure except that a single metal ion is bound to the Mβ-site (pdb code: 1o12). The metal ligation scheme in the NagA from E. coli must be significantly different since the H×H motif from the end of β-stand 1 is replaced by Q×N and thus it is highly likely that this enzyme can bind only a single divalent cation in the active site. This fundamental divergence in the active sites of NagA from these sources suggests that the mechanism for the activation of the hydrolytic water and substrate required for catalysis must differ to a significant degree. NagA has been characterized as a member of the amidohydrolase superfamily based on an exhaustive set of sequence and structural comparisons (8, 9, 11). This superfamily is a diverse group of enzymes that catalyzes the hydrolytic cleavage of amide bonds to nucleic acids, amino acids, and sugars (8, 9). Within this superfamily, four metal ligation schemes have been identified. The most prevalent is a binuclear metal center where the two divalent cations are bridged by a hydroxide from solvent. An additional bridging ligand from the protein may include a carboxylated lysine from the end of β-strand 4, a glutamate from the end of β-strand three or four, or a cysteine from the end of β-strand 2 (8, 9). Enzymes of this type include dihydroorotase (12), the phosphotriesterase homology protein (13), renal dipeptidase (14) and d-amino acid deacetylase (15). Many members of the amidohydrolase superfamily bind a single divalent cation, including cytosine (16) and adenosine deaminase (17). This class of enzymes binds one metal at the α-position via ligation to the H×H motif from β-strand 1, a histidine from the end of β-strand 5 and an invariant asparate from β-strand 8. The third ligation scheme, which may include NagA from E. coli and T. maritima, utilizes a single metal bound exclusively to the β-site. The most extreme example within the amidohydrolase superfamily is uronate isomerase from E. coli. This enzyme catalyzes the isomerization of d-glucuronate to d-fructuronate and a metal ion is not required for catalytic activity (18). There are a significant number of issues regarding the mechanism for substrate hydrolysis by NagA that are unresolved. It is unclear how the divalent cation binds within the active site of NagA and how the water molecule is activated for nucleophilic attack on the amide bond. The disparity in the metal ligation schemes between the E. coli and B. subtilis forms of NagA suggests a divergence in the evolution of this enzyme at a significant locale within the active site. In this paper, we have systematically interrogated the mechanism of substrate hydrolysis by NagA from E. coli with a battery of substrate analogs and active site mutants using a host of metal-substituted forms of the enzyme. We conclude from these studies that NagA from E. coli can bind up to one divalent cation for the activation of the hydrolytic water molecule. This water molecule is further activated by general base catalysis through the abstraction of a proton by Asp-273. There is no evidence to suggest that Glu-131 plays any role in catalysis other than to help coordinate the single divalent cation. The enzyme is rate limited by the cleavage of the amide bond.