Probing the Catalytic Mechanism of Vibrio harveyi GH20 β-N-Acetylglucosaminidase by Chemical Rescue

Abstract

Background: Vibrio harveyi GH20 β-N-acetylglucosaminidase (VhGlcNAcase) is a chitinolytic enzyme responsible for the successive degradation of chitin fragments to GlcNAc monomers, activating the onset of the chitin catabolic cascade in marine Vibrios.

Methods: Two invariant acidic pairs (Asp303-Asp304 and Asp437-Glu438) of VhGlcNAcase were mutated using a site-directed mutagenesis strategy. The effects of these mutations were examined and the catalytic roles of these active-site residues were elucidated using a chemical rescue approach. Enhancement of the enzymic activity of the VhGlcNAcase mutants was evaluated by a colorimetric assay using pNP-GlcNAc as substrate.

Results: Substitution of Asp303, Asp304, Asp437 or Glu438 with Ala/Asn/Gln produced a dramatic loss of the GlcNAcase activity. However, the activity of the inactive D437A mutant was recovered in the presence of sodium formate. Our kinetic data suggest that formate ion plays a nucleophilic role by mimicking the β-COO-side chain of Asp437, thereby stabilizing the reaction intermediate during both the glycosylation and the deglycosylation steps.

Conclusions: Chemical rescue of the inactive D437A mutant of VhGlcNAcase by an added nucleophile helped to identify Asp437 as the catalytic nucleophile/base, and hence its acidic partner Glu438 as the catalytic proton donor/acceptor.

General significance: Identification of the catalytic nucleophile of VhGlcNAcases supports the proposal of a substrate-assisted mechanism of GH20 GlcNAcases, requiring the catalytic pair Asp437-Glu438 for catalysis. The results suggest the mechanistic basis of the participation of β-N-acetylglucosaminidase in the chitin catabolic pathway of marine Vibrios.

Fig 1. Comparison of the modelled structure of VhGlcNAcase with other GlcNAcases.

(A) Multiple sequence alignment of GH20 glycoside hydrolases. The amino acid sequence of Vibrio harveyi β-N-acetylglucosaminidase, VhGlcNAcase (SwissProt: D9ISE0) was retrieved from the Uniprot database. This sequence was aligned with those of Serratia marcescens chitobiase, SmCHB (SwissProt: Q54468), Streptomyces plicatus β-N-acetylhexosaminidase, SpHex (SwissProt: O85361), Paenibacillus sp. β-hexosaminidase, PsHex1T (SwissProt: D2KW09), human β-hexosaminidase A (α-chain), HsHexA (SwissProt: P06865) and human β-hexosaminidase B (β-chain), HsHexB (SwissProt: P07686). The putative amino acid residues that are important for GlcNAcase activity are indicated with blue stars. (B) Surface representation of the active-site pocket of VhGlcNAcase (in blue) docked with GlcNAc₂ (in yellow stick) from SmCHB (PDB entry: 1QBB). The solvent-accessible surface of D437-E438 is highlighted in pink and the buried surface of D303-D304 is highlighted in green. (C) Superimposition of both conserved acidic pairs (Asp303-Asp304 and Asp437-Glu438) of modelled VhGlcNAcase (in magenta stick) with the crystal structure of SmCHB (in green stick) in complex with GlcNAc₂. N atoms are shown in blue and O atoms in red.

Fig 2. Activity/pH profiles of VhGlcNAcase and its mutant D437A.

The specific activity of VhGlcNAcase (solid line, left y axis) and the mutant D437A (dashed line, right y axis) was measured at pH = 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 in the McIlvaine’s sodium phosphate-citric acid buffer system. pNP-GlcNAc was used as substrate and the reaction was carried out for 10 min at 37°C.

Fig 3. Effect of sodium formate on pNP-GlcNAc hydrolysis by VhGlcNAcase and its mutants.

Various concentrations of sodium formate (0.0–2.0 M) were added to the reaction mixture, which contained 500 μM pNP-GlcNAc and 100 mM sodium phosphate buffer, pH 7.0, at 37°C. v/v₀ is the fractional activity of the enzyme, i.e. activity in the presence of sodium formate relative to that in its absence. (A) The D437A (filled diamonds) and D437N (open inverted triangles) mutants. (B) The mutants E438A (filled squares) and E438Q (open triangles). The wild-type VhGlcNAcase (open circles) are shown in both A and B.

Fig 4

(A) Time-courses of reactions of the D437A mutant with and without sodium formate. Reaction mixtures (200 μL), containing 2 μg of D437A mutant and 500 μM of pNP-GlcNAc and varied concentrations of sodium formate (0, 0.1, 0.25, 0.5, 1.0, and 2.0 M) and 100 mM sodium phosphate buffer, pH 7.0, were incubated at 37°C for 0–60 min, and the reaction terminated with 100 μL of 3 M Na₂CO₃. Release of pNP, monitored at A₄₀₅, was converted to molar quantities using a calibration curve of pNP (0–20 nmol). The linear part of the reaction progress was shown as an inset. (B) Initial reaction rates for the mutant D437A of VhGlcNAcase in the presence of sodium formate were obtained from Michaelis-Menten plots. Reaction rates were measured using pNP-GlcNAc (0–500 μM) as the substrate, 5 μg of the mutant D437A of VhGlcNAcase and sodium formate at the same range of concentraitons as described above. (C) Activation by formate anion was evaluated by means of Lineweaver-Burk plots of initial reaction rates.

Fig 5. Chemical recue of the D437A mutant by sodium formate.

Three kinetic constant ratios: the apparent first-order rate constants, k_cat (open circles), apparent Michaelis constants, K_m (open squares) and apparent second-order rate constants, k_cat/K_m (open triangles), were plotted as a function of sodium formate concentration.

Fig 6

(A) Proposed mechanism of formate-mediated chemical rescue of the activity of the VhGlcNAcase D437A mutant. Formate ion is involved in both the glycosylation and deglycosylation steps by providing charge stabilization of transition states that flank the oxazolinium ion. (B) Proposed mechanism of azide-mediated chemical rescue with the SpHex D313A mutant. Azide ion is involved only in the deglycosylation step, acting to open the cyclic oxazolinium ion intermediate [21]. Hydroxyl groups and C₆ have been omitted for clarity.