Structural and functional characterization of the Clostridium perfringens N-acetylmannosamine-6-phosphate 2-epimerase essential for the sialic acid salvage pathway

Abstract

Pathogenic bacteria are endowed with an arsenal of specialized enzymes to convert nutrient compounds from their cell hosts. The essential N-acetylmannosamine-6-phosphate 2-epimerase (NanE) belongs to a convergent glycolytic pathway for utilization of the three amino sugars, GlcNAc, ManNAc, and sialic acid. The crystal structure of ligand-free NanE from Clostridium perfringens reveals a modified triose-phosphate isomerase (β/α)8 barrel in which a stable dimer is formed by exchanging the C-terminal helix. By retaining catalytic activity in the crystalline state, the structure of the enzyme bound to the GlcNAc-6P product identifies the topology of the active site pocket and points to invariant residues Lys(66) as a putative single catalyst, supported by the structure of the catalytically inactive K66A mutant in complex with substrate ManNAc-6P. (1)H NMR-based time course assays of native NanE and mutated variants demonstrate the essential role of Lys(66) for the epimerization reaction with participation of neighboring Arg(43), Asp(126), and Glu(180) residues. These findings unveil a one-base catalytic mechanism of C2 deprotonation/reprotonation via an enolate intermediate and provide the structural basis for the development of new antimicrobial agents against this family of bacterial 2-epimerases.

Keywords: 1H NMR Spectroscopy; Carbohydrate; Enzyme Mechanism; Mutagenesis; Sialic Acid; Sugar 2-Epimerase; Sugar Phosphate; X-ray Crystallography.

FIGURE 1.

Overall structure of NanE. A and B, orthogonal views of the CpNanE structure, colored according to the type of secondary structures. The dominant α-β barrel structure hosts two chlorine ions, one (labeled Cl2) in the active site and the other (Cl1) at the periphery of the α/β barrel. C, domain-swapped dimeric assembly showing the interchange of the C-terminal helices. Secondary structure elements are numbered α0 to α8 and β1 to β8. D, electrostatic potential mapped on the molecular surface of a subunit of CpNanE^wt in complex with GlcNAc-6P is shown at −5 kT/e (red) to +5 kT/e (blue). The surface was calculated with APBS (53) and drawn using the Python Molecular Viewer program (54).

FIGURE 2.

Electron density maps in the active center. A, stereoviews, shown in divergent (“walleyed”) mode, of bound (A) GlcNAc-6P product in the active site of CpNanE^wt, showing catalysis in the crystal and (B) ManNAc-6P substrate in the active site of the CpNanE^K66A mutant. The maximum likelihood/σA weighted 2F_obs − F_calc electron densities are shown at 1.0 and 0.5 electrons/Ų for GlcNAc-6P and ManNAc-6P, respectively. Figures were drawn using PyMOL (55). C, schematic diagram of the active center of CpNanE with bound GlcNAc-6P and interacting side chains. Water molecules are shown as small shaded spheres. Hydrogen bonds and electrostatic interactions are shown by dotted lines and wavy lines, respectively, and some inter-atomic distances (Å) are indicated using arrows for clarity. Figure was drawn with CHEMDRAW (CambridgeSoft Corporation, Cambridge, MA).

FIGURE 3.

Sequence alignment of representative NanE proteins. The primary sequences of NanE proteins from C. perfringens (Clos_perf), Candidatus arthromitus (Cand_arth), Enterococcus pallens (Ente_pall), B. fragilis (Bact_frag), S. pneumoniae (Strep_pneu), Bacillus coagulans (Baci_coag), and Anaerobiospirillum succiniciproducens (Anae_succ) are aligned. The Clos_perf sequence corresponds to the gene product that has been used in this study. Secondary structure elements are labeled and shown above the alignment. The four invariant residues that have been mutated are shown with a gray star. The figure was generated using MultAlin and Esprit (56, 57).

FIGURE 4.

Real time ¹H NMR analysis of CpNanE activity. A, time course of ManNAc-6P (2 mm) epimerization by CpNanE in the presence of β-alanine (1 mm) as an internal standard was monitored. Selected regions of the spectra acquired between 0 and 34 min are shown. B, overlay of the spectral region from 2.2 to 2.3 ppm showing the disappearance of the characteristic peak for the ManNAc-6P methyl resonances versus appearance of GlcNAc-6P methyl resonance. C, initial velocities of ManNAc-6P epimerization by CpNanE in the presence of different concentrations of ManNAc-6P. A Michaelis-Menten curve was fitted using the formula: v/[E]₀ = k_cat [S]/(K_m + [S]).

FIGURE 5.

Proposed catalytic mechanism of CpNanE. A, once the C2 proton is abstracted by the base catalyst Lys⁶⁶, a negatively charged intermediate is stabilized by the salt bridge and Glu¹⁸⁰. Presence of the Asp¹²⁶-Arg⁴³ salt bridge in proximity of the bound ManNAc-6P substrate reinforces the reactivity of Lys⁶⁶. A proton is donated by the same Lys⁶⁶ catalyst to the enolate intermediate to yield the GlcNAc-6P product. B, a one-base mechanism for the interconversion of ManNAc-6P and GlcNAc-6P requires that Lys⁶⁶ adjust its position in response to the rotation of the 1-keto intermediate around the C2-C3 bond. Yellow dotted arrows indicate the concerted motions. The figure was drawn with PyMOL (55).