The Functional Unit of Neisseria meningitidis 3-Deoxy-ᴅ-Arabino-Heptulosonate 7-Phosphate Synthase Is Dimeric

Abstract

Neisseria meningitidis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (NmeDAH7PS) adopts a homotetrameric structure consisting of an extensive and a less extensive interface. Perturbation of the less extensive interface through a single mutation of a salt bridge (Arg126-Glu27) formed at the tetramer interface of all chains resulted in a dimeric DAH7PS in solution, as determined by small angle X-ray scattering, analytical ultracentrifugation and analytical size-exclusion chromatography. The dimeric NmeDAH7PSR126S variant was shown to be catalytically active in the aldol-like condensation reaction between D-erythrose 4-phosphate and phosphoenolpyruvate, and allosterically inhibited by L-phenylalanine to the same extent as the wild-type enzyme. The dimeric NmeDAH7PSR126S variant exhibited a slight reduction in thermal stability by differential scanning calorimetry experiments and a slow loss of activity over time compared to the wild-type enzyme. Although NmeDAH7PSR126S crystallised as a tetramer, like the wild-type enzyme, structural asymmetry at the less extensive interface was observed consistent with its destabilisation. The tetrameric association enabled by this Arg126-Glu27 salt-bridge appears to contribute solely to the stability of the protein, ultimately revealing that the functional unit of NmeDAH7PS is dimeric.

Fig 1. A). Quaternary structure of NmeDAH7PS (PDB code: 4HSN) indicating the tight dimer interface and tetramer interface. The core catalytic (β/α)₈ barrel is coloured blue and the N-terminal and internal extensions are coloured red and yellow, respectively. Manganese metal ion (purple), ligand (PEP) and Glu27-Arg126 salt bridge (green-sticks) A schematic of secondary structural features of monomeric NmeDAH7PS according to residue number, circles denote β-sheets and rectangles denote α-helices B).

Hydrogen bonding (red) and salt bridge (blue) interactions identified by analysis of each interface. Top. Tight-dimer. Bottom. Tetramer C) Hydrophobic interactions (green) at each interface of NmeDAH7PS. Top. Tight-dimer. Bottom. Tetramer. Chain B, C and D are denoted `, “and “`respectively.

Fig 2. (A) Analytical SEC traces of NmeDAH7PS^WT and NmeDAH7PS^R126S at two different concentrations. NmeDAH7PS^WT 1 mg/mL (red) and 0.2 mg/mL (orange). NmeDAH7PS^R126S 1 mg/mL (blue) and 0.2 mg/mL (light blue). B. Calculated sedimentation coefficient c(s) distribution plot of wild-type NmeDAH7PS (top) and NmeDAH7PS^R126S (bottom). For both enzymes, the distribution of species for three different concentrations are shown: 0.2 mg/mL (red), 0.4 mg/mL (green) and 1.2 mg/mL (blue). The sedimentation velocity data, fit data and residuals from the fits are shown in S2 Fig.

Fig 3. SAXS scattering profiles collected for NmeDAH7PS^WT and NmeDAH7PS^R126S.

(A) Overlay of the scattering data of NmeDAH7PS^WT (closed circles) and NmeDAH7PS^R126S variant (open circles) and (B) CRYSOL fits of the NmeDAH7PS^R126S variant data to the theoretical scattering data generated from the R126S tetramer (dotted line) and tight-dimer (dashed line). The discrepancies of the fit of the theoretical scattering of tetrameric and dimeric forms to the experimentally determined data (χ) were calculated as 22.80 and 0.67 respectively.

Fig 4. Catalytic activity of NmeDAH7PS^R126S (blue) NmeDAH7PS^WT (red) over 24 hour time period relative to maximum activity observed for each enzyme at the beginning of the experiment.

Fig 5. A. Effect of the aromatic amino acids, Phe (black), Tyr (red) and Trp (blue) on the catalytic activity of NmeDAH7PS^R126S (open circles) in comparison to data previously reported for the wild-type enzyme (closed circles) [12]. B. Isothermal titration calorimetry binding isotherm for NmeDAH7PS^R126S fit to a two-site sequential binding model.

Fig 6. Tetramer interface of NmeDAH7PS^WT (green) (PDB code:4HSN) and NmeDAH7PS^R126S (cyan) indicating the variable positioning of Tyr26 in place of the conserved water molecule from the NmeDAH7PS^WT.

The loss of salt bridge between Glu27 and Arg126 for the NmeDAH7PS^R126S variant is also illustrated.