Substrate binding modes and anomer selectivity of chitinase A from Vibrio harveyi

Abstract

High-performance liquid chromatography mass spectrometry (HPLC MS) was employed to assess the binding behaviors of various substrates to Vibrio harveyi chitinase A. Quantitative analysis revealed that hexaNAG preferred subsites -2 to +2 over subsites -3 to +2 and pentaNAG only required subsites -2 to +2, while subsites -4 to +2 were not used at all by both substrates. The results suggested that binding of the chitooligosaccharides to the enzyme essentially occurred in compulsory fashion. The symmetrical binding mode (-2 to +2) was favored presumably to allow the natural form of sugars to be utilized effectively. Crystalline alpha chitin was initially hydrolyzed into a diverse ensemble of chitin oligomers, providing a clear sign of random attacks that took place within chitin chains. However, the progressive degradation was shown to occur in greater extent at later time to complete hydrolysis. The effect of the reducing-end residues were also investigated by means of HPLC MS. Substitutions of Trp275 to Gly and Trp397 to Phe significantly shifted the anomer selectivity of the enzyme toward beta substrates. The Trp275 mutation modulated the kinetic property of the enzyme by decreasing the catalytic constant (k (cat)) and the substrate specificity (k (cat)/K (m)) toward all substrates by five- to tenfold. In contrast, the Trp397 mutation weakened the binding strength at subsite (+2), thereby speeding up the rate of the enzymatic cleavage toward soluble substrates but slowing down the rate of the progressive degradation toward insoluble chitin.

Fig. 1

The active site of V. harveyi chitinase A mutant E315M bound to hexaNAG. a A surface representation of hexaNAG that fully occupied subsites −4 to +2 within the substrate binding cleft of the enzyme. The sugar rings of NAG₆ are shown in sticks. b A stick model of the binding cleft of chitinase A mutant E315M complexed with NAG₆. N atoms are shown in blue and O atoms in red. C atoms are in marine blue for the amino acid residues and in yellow for the sugar residues. The cleavage site is indicated by an arrow, and Trp275 and Trp397 are represented in blue. The structure of E315M + NAG₆ complex is obtained from the PDB data base (PDB code, 3B9A) [24] and displayed by the program PyMol (http://www.pymol.org/)

Fig. 2

Three possible models of chitin oligosaccharide bindings to the multiple binding subsites of V. harveyi chitinase A. Hydrolysis of a NAG₅, b NAG₆, and c crystalline α chitin. NAG unit with β configuration is shown in black circle, and NAG residue with α or β configuration with an equilibrium ratio is shown in gray circle. Types of the reducing-end anomers are predicted based on the retaining mechanism as suggested for family-18 chitinases

Fig. 3

An HPLC MS profile of the initial products obtained from partial hydrolysis of NAG6 by wild-type chitinase A. The hydrolytic reaction (50 μL) was carried out on ice to minimize the rate of mutarotation. After 3 min, aliquots of 10 μL of the reaction mixture were subjected to HPLC MS analysis

Fig. 4

Partial hydrolysis of crystalline α chitin by the wild-type chitinase A. a Time course of chitin hydrolysis. The hydrolytic reactions were carried out at on ice (0 °C) from 0 to 180 min and were analyzed immediately by HPLC MS. Molar concentrations of the hydrolytic products were estimated using the standard curves of NAG_1–6. b The cleavage ratios of NAG₂/NAG_3–6 were calculated based on the molar concentration of each corresponding sugar. The reaction products generated during 0–10 min are indicated as an inset. Open circle dimers, open square trimers, open triangle tetramers; filled circle pentamers, filled square and hexamers

Fig. 5

Time-course of substrate consumption by chitinase A. Hydrolysis of NAG₅ by a wild type, b mutant W275G, and c mutant W397F. The hydrolytic reactions carried out at on ice (0 °C) from 0 to 155 min were analyzed by HPLC ESI/MS as described in texts. Rate of β anomer consumption is presented by a black line and rate of β anomer consumption by a broken line

Fig. 6

Plausible effects of point mutation on the substrate binding preference and the anomer selectivity. Binding of NAG₅ to a wild-type chitinase, b mutant W275G, and c mutant W397F. The β configuration is shown in black circle, NAG residue with α or β configuration with an equilibrium ratio is shown in gray circle, and NAG residue with α or β configuration that binds to a loosen-affinity binding site is shown in gray-filled in dark circle. The cleavage site is indicated by an arrow