Crystal structures of Vibrio harveyi chitinase A complexed with chitooligosaccharides: Implications for the catalytic mechanism

Abstract

This research describes four X-ray structures of Vibrio harveyi chitinase A and its catalytically inactive mutant (E315M) in the presence and absence of substrates. The overall structure of chitinase A is that of a typical family-18 glycosyl hydrolase comprising three distinct domains: (i) the amino-terminal chitin-binding domain; (ii) the main catalytic (α/β)₈ TIM-barrel domain; and (iii) the small (α + β) insertion domain. The catalytic cleft of chitinase A has a long, deep groove, which contains six chitooligosaccharide ring-binding subsites (−4)(−3)(−2)(−1)(+1)(+2). The binding cleft of the ligand-free E315M is partially blocked by the C-terminal (His)₆-tag. Structures of E315M-chitooligosaccharide complexes display a linear conformation of pentaNAG, but a bent conformation of hexaNAG. Analysis of the final 2F_o − F_c omit map of E315M-NAG6 reveals the existence of the linear conformation of the hexaNAG at a lower occupancy with respect to the bent conformation. These crystallographic data provide evidence that the interacting sugars undergo conformational changes prior to hydrolysis by the wild-type enzyme.

Introduction

Chitin, a highly stable homopolysaccharide of β (1 → 4)-linked N-acetyl-d-glucosamine (GlcNAc or NAG)¹ is widely distributed in the shells of crustaceans; the cuticles of insects; the shells and skeletons of molluscs; and the cell walls of fungi. Chitin degradation is of considerable interest because the products have potential applications in the fields of biomedicine, agriculture, nutrition, and biotechnology. Chitinases (EC 3.2.1.14) are major enzymes that hydrolyse chitin into oligosaccharide fragments. These enzymes are found in organisms that possess chitin as a constituent or that use it as a nutrient source. Bacteria produce chitinases in order to utilise chitin as a source of carbon and nitrogen (Bhattacharya et al., 2007, Keyhani and Roseman, 1999). Fungal chitinases play a similar nutritional role but are additionally involved in fungal development and morphogenesis (Kuranda and Robbins, 1991, Sahai and Manocha, 1993). Plants produce chitinases as a defence mechanism against pathogenic fungi (Leah et al., 1991). Animal chitinases are involved in dietary uptake processes (Jeuniaux, 1961). Human chitinases are responsible for hyperresponsiveness and inflammation of the airways of allergic asthma patients (Donnelly and Barnes, 2004, Kawada et al., 2007).

The carbohydrate active enzyme (CAZy) database (http://www.cazy.org/) classifies carbohydrate enzymes into functional families (glycosyl hydrolases, glycosyl transferases, polysaccharide lyases, carbohydrate esterases, and carbohydrate-binding modules), which are further subdivided into structurally related families designated by number. Following this classification, chitinases are listed as GH family-18 and GH family-19. These two families show no homology in both structure and mechanism. The catalytic domain of family-18 chitinases consists of an (α/β)₈-barrel with a deep substrate-binding cleft formed by loops following the C-termini of the eight parallel β-strands (Fukamizo, 2000, Hollis et al., 2000, Perrakis et al., 1994, Terwisscha van Scheltinga et al., 1996). In contrast, the catalytic domain of family-19 chitinases comprises two lobes, each of which is rich in α-helical structure (Hart et al., 1995). Family-18 chitinases are known to catalyse the hydrolytic reaction through the ‘substrate-assisted’ or’retaining mechanism’ (Brameld and Goddard, 1998a, Terwisscha van Scheltinga et al., 1995, Tews et al., 1997), whereas family 19 chitinases employ the ‘single displacement’ or ’inversion mechanism’ (Brameld and Goddard, 1998b). The catalytic mechanism of family-18 chitinases involves protonation of the leaving group by an absolutely conserved glutamic acid (equivalent to Glu315 of Serratia marcescens chitinase A), followed by substrate distortion into a ‘boat’ conformation at subsite −1 and the stabilisation of an oxazolinium intermediate by the sugar N-acetamido group. The resultant bond cleavage yields the retention of anomeric configuration in the products (Armand et al., 1994, Fukamizo et al., 2001, Honda et al., 2004, Sasaki et al., 2002).

Chitinases from both families are further divided into exo- and endochitinases. Exochitinase activity represents a progressive action that starts at the non-reducing end of a chitin chain and successively releases diacetylchitobiose (NAG2) units, where as, endochitinase activity involves random cleavage at internal points within a chitin chain (Robbins et al., 1988). The active sites of family-18 endochitinases, such as S. marcescens chitinase A and Hevea brasiliensis chitinase (hevamine), are groove-like structures with openings above and at both ends (Brameld and Goddard, 1998a, Hart et al., 1995). In contrast, the active sites of exochitinases, such as S. marcescens ChiB, have tunnel-like morphologies (Van Aalten et al., 2001).

We previously isolated the Chi A gene encoding the 95-kDa chitinase precursor (GenBank Accession No: Q9AMP1) from the genome of Vibrio carchariae type strain LMG7890. Based on genotypical and phenotypical features analysed by Pedersen et al. (1998), V. carchariae has been re-classified as a heterotypic synonym of Vibrio harveyi. To follow the new systematic taxonomy, V. carchariae will be referred to as V. harveyi in this and later studies. V. harveyi (formerly V. carchariae) is a marine bacterium that secretes high levels of a 63-kDa endochitinase A (Suginta et al., 2000). This family-18 glycosyl hydrolase shows greatest affinity towards chitohexamer, which suggests the substrate-binding cleft of this enzyme that comprises an array of six GlcNAc-binding sites (Suginta et al., 2005), comparable to that of S. marcescens Chi A and hevamine (Papanikolau et al., 2001, Perrakis et al., 1994, Terwisscha van Scheltinga et al., 1996). The gene that encodes chitinase A without the 253-aa C-terminal proprotein fragment was subsequently cloned and functionally expressed in Escherichia coli (Suginta et al., 2004). Substitution of Glu315 to Met/Gln completely abolished the hydrolysing activity against soluble and insoluble substrates, confirming that this residue is essential for catalysis (Suginta et al., 2005).

In this study, we describe the crystal structures of V. harveyi chitinase A and its catalytically inactive E315M mutant, as well as the E315M mutant soaked with NAG5 and NAG6. The overall structures of E315M with and without substrates are almost identical to the wild-type structure. However, the relative conformations of NAG5 and NAG6 bound to E315M are very different. These static structures provide hints to the conformational changes in chitooligosaccharide conformation during binding and hydrolysis, allowing for a catalytic mechanism of the enzyme to be proposed.