Structural and functional determination of homologs of the Mycobacterium tuberculosis N-acetylglucosamine-6-phosphate deacetylase (NagA)

Abstract

The Mycobacterium tuberculosis (Mtb) pathogen encodes a GlcNAc-6-phosphate deacetylase enzyme, NagA (Rv3332), that belongs to the amidohydrolase superfamily. NagA enzymes catalyze the deacetylation of GlcNAc-6-phosphate (GlcNAc6P) to glucosamine-6-phosphate (GlcN6P). NagA is a potential antitubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for Mtb cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments. Here, we report the structural and functional characterization of NagA from Mycobacterium smegmatis (MSNagA) and Mycobacterium marinum (MMNagA), close relatives of Mtb Using a combination of X-ray crystallography, site-directed mutagenesis, and biochemical and biophysical assays, we show that these mycobacterial NagA enzymes are selective for GlcNAc6P. Site-directed mutagenesis studies revealed crucial roles of conserved residues in the active site that underpin stereoselective recognition, binding, and catalysis of substrates. Moreover, we report the crystal structure of MSNagA in both ligand-free form and in complex with the GlcNAc6P substrate at 2.6 and 2.0 Å resolutions, respectively. The GlcNAc6P complex structure disclosed the precise mode of GlcNAc6P binding and the structural framework of the active site, including two divalent metals located in the α/β binuclear site. Furthermore, we observed a cysteine residue located on a flexible loop region that occludes the active site. This cysteine is unique to mycobacteria and may represent a unique subsite for targeting mycobacterial NagA enzymes. Our results provide critical insights into the structural and mechanistic properties of mycobacterial NagA enzymes having an essential role in amino-sugar and nucleotide metabolism in mycobacteria.

Keywords: Mycobacterium tuberculosis; N-acetylglucosamine-6-phosphate deactylase; TB; X-ray crystallography; bacterial cell wall; carbohydrate; crystal structure; enzyme kinetics; peptidoglycan; tuberculosis.

Figure 1.

Schematic diagram of the pathways of amino sugar metabolism in M. tuberculosis. The reaction catalyzed by NagA (GlcNAc6P deacetylase) is highlighted in red. Glc6P, glucose-6-phosphate; F6P, fructose-6-phosphate; GlcN6P, glucosamine-6-phosphate; GlcN1P, glucosamine-1-phosphate; GlcNAc1P, GlcNAc-1-phosphate; SugI, integral sugar transporter; Pgi, glucose-6-phosphate isomerase; GlmS, glutamine-fructose-6-phosphate aminotransferase; GlmM, phosphoglucosamine mutase; GlmU, bifunctional acetyltransferase/uridyltransferase.

Figure 2.

The NagA operon in M. tuberculosis. The operon organization was taken from Xbase. The accession numbers for these genes in M. tuberculosis H37Rv are as follows: Rv3329 (probable aminotransferase), Rv3330 (dacB1, probable penicillin-binding protein d-alanyl-d-alanine carboxypeptidase), Rv3331 (sugI, probable sugar-transport integral membrane protein), and Rv3332 (nagA, GlcNAc-6-phosphate deacetylase).

Figure 3.

Panel of carbohydrates probed in the kinetic studies.

Figure 4.

Substrate dependence of MMNagA activity. Michaelis–Menten curves were fitted, and selected curves are shown for MMNagA with the substrates GlcNAc6P (A), GalNAc6P (B), ManNAc6P (C), and GlcNAc6S (D). The initial velocity data were plotted against the substrate concentration, and each assay was carried out in triplicate and expressed as a value ± standard error of mean.

Figure 5.

Crystal structure of MSNagA. Shown is MSNagA structure with one subunit (chain A) represented as a cartoon and the other subunit (chain B) with surface representation. Domain I is colored blue, and domain II is colored brown (chain A)/red (chain B)). The metal ions are represented as gray spheres, and the GlcNAc6P ligand is shown in stick representation.

Figure 6.

The GlcNAc6P substrate-binding site in MSNagA. A, illustration showing GlcNAc6P with green carbon atoms, the metal ion as a pale-yellow sphere, and selected amino acid residues in stick representation (colored gray for chain A, and magenta for chain B). B, schematic diagram of the interactions of MSNagA with GlcNAc6P. The residues that interact with the M1 metal are shown in green, the residues that interact with the M2 metal are shown in red, the residues that interact with the GlcNAc6P substrate are shown in black, the residues that interact with the GlcNAc6P substrate from the opposing MSNagA monomer are shown in blue, and the Asp-267 residue that is mutated in the ligand-bound structure is shown in purple.

Figure 7.

Structure of the metal-binding site of MSNagA. A, illustration showing metal-binding site in apo-MSNagA structure (chain A, magenta) superposed with ligand-bound structure (chain A, blue). Silver spheres, Zn²⁺; red spheres, H₂O. GlcNAc6P with green carbon atoms and selected amino acid residues are shown in stick representation (colored by apo (magenta) or ligand-bound (blue)). B, superposition of the metal-binding site of chain A and chain B of the ligand-bound structure. Light blue, chain A; light brown, chain B; silver spheres, Zn²⁺; orange sphere, Cd²⁺; green sphere, Cl⁻. Selected amino acid residues are shown in stick representation (colored by chain A (blue) or ligand-bound (salmon)).