Structure of the D142N mutant of the family 18 chitinase ChiB from Serratia marcescens and its complex with allosamidin

https://doi.org/10.1016/j.bbapap.2003.09.014Get rights and content

Abstract

Catalysis by ChiB, a family 18 chitinase from Serratia marcescens, involves a conformational change of Asp142 which is part of a characteristic D140XD142XE144 sequence motif. In the free enzyme Asp142 points towards Asp140, whereas it rotates towards the catalytic acid, Glu144, upon ligand binding. Mutation of Asp142 to Asn reduced kcat and affinity for allosamidin, a competitive inhibitor. The X-ray structure of the D142N mutant showed that Asn142 points towards Glu144 in the absence of a ligand. The active site also showed other structural adjustments (Tyr10, Ser93) that had previously been observed in the wild-type enzyme upon substrate binding. The X-ray structure of a complex of D142N with allosamidin, a pseudotrisaccharide competitive inhibitor, was essentially identical to that of the wild-type enzyme in complex with the same compound. Thus, the reduced allosamidin affinity in the mutant is not caused by structural changes but solely by the loss of electrostatic interactions with Asp142. The importance of electrostatics was further confirmed by the pH dependence of catalysis and allosamidin inhibition. The pH-dependent apparent affinities for allosamidin were not correlated with kcat, indicating that it is probably better to view the inhibitor as a mimic of the oxazolinium ion reaction intermediate than as a transition state analogue.

Introduction

Chitinases catalyse the hydrolysis of chitin, a biopolymer of β(1–4)-linked N-acetylglucosamine. These enzymes belong to two families of glycoside hydrolases, namely family 18 and family 19 [1]. The catalytic domains of family 18 chitinases have a TIM-barrel ((βα)8 barrel) fold. The β-strand four of the TIM-barrel contains a characteristic DXDXE sequence motif that includes the glutamate residue that protonates the oxygen in the scissile glycosidic bond [2], [3], [4], [5], [6]. One well-studied member of this family of enzymes is Chitinase B (ChiB) from the soil bacterium Serratia marcescens. ChiB is an exochitinase that degrades chitin chains from their non-reducing ends [7], [8]. In addition to the catalytic domain, this enzyme has a small chitin-binding domain that extends the substrate-binding cleft towards the reducing end of the polysaccharide chain [8].

Catalysis in family 18 chitinases involves the N-acetyl group of the sugar bound in the −1 subsite of the enzyme ([2], [3], [4], [5], [6]; Fig. 1). Protonation of the glycosidic bond and leaving group departure are accompanied by nucleophilic attack by the carbonyl oxygen of the N-acetyl group on the anomeric carbon, thus yielding an oxazolinium ion intermediate ([2], [3], [4], [5], [6]; Figs. 1C and 2A). The formation of this type of intermediate has also been described for N-acetyl-β-hexosaminidases [9], [10]. Catalysis in family 18 chitinases involves conserved residues in the DXDXE motif, which in case of ChiB are the catalytic acid, Glu144, as well as Asp142 and Asp140 [6], [11]. Structural studies have shown that the side chain of Asp142 (and its equivalents in other family 18 chitinases) may occur in two conformations, either pointing “down” into the TIM barrel (towards Asp140) or pointing “up” towards Glu144 (Fig. 1; [5], [6], [11], [12], [13]). Binding of substrate fixes the Asp142 side chain in the “up” position, which is of importance for substrate-distortion and catalysis (see Fig. 1 and below).

Asp142 is important for catalysis since its mutation to alanine in ChiB (see below) and other family 18 chitinases [12] abolishes enzyme activity. Replacement with Asn also reduces activity, but to a lesser extent [6], [14], [15]. The D142N mutant is interesting because its considerable residual activity indicates that catalysis still proceeds via a wild-type-like mechanism. On the other hand, this mutant and an analogous mutant in another family 18 chitinase [14] show distinct changes in their pH–activity profiles.

To gain further insight into the contribution of Asp142 to catalysis and to study possible structural effects of the D142N mutation we have determined the crystal structure of the ChiB mutant. In addition, enzymological and structural studies have been conducted with allosamidin, a pseudotrisaccharide that is known to inhibit family 18 chitinases [5], [6], [11], [16]. Allosamidin is structurally similar to the proposed reaction intermediate (Fig. 2) and its high affinity for family 18 chitinases has led to the suggestion that the inhibitor is a transition state analogue [3], [4], [5], [6], [11], [17].

Section snippets

Mutagenesis, protein purification and crystallization

The ChiB D142N mutant was produced by site directed mutagenesis using the Stratagene QuickChange kit (Stratagene, La Jolla, CA) and was overexpressed and purified as previously described [7]. The pure protein was lyophilised, dissolved to 1.0 mg/ml in 25 mM Tris–HCl, pH 8.0, dialyzed overnight in the same buffer and concentrated to 10 mg/ml with Ultrafree-MC concentration tubes with a 10,000 MW cut-off (Millipore, Bedford, MA, USA). Subsequently, hanging drop vapour diffusion experiments were

The D142N structure

The D142N structure was refined to 1.85 Å with R-factors converging to R=0.159 and Rfree=0.194 (Table 1). The structure was similar to the structure of the wild-type apo-enzyme. Superposition of the B monomers yielded an RMSD of 0.484 Å (Cα's only), a value that is dominated by concerted motions of areas peripheral to the active site. Conspicuous differences were observed for several side chain conformations in the active site region (Fig. 3). Compared to Asp142 in the wild-type, the side chain

Discussion

The only structural differences between wild-type ChiB and the D142N mutant that seem to affect catalysis concern the position of residue 142 and simultaneous adjustments of Tyr10 and Ser93 (Fig. 3). The adjustments of residues 10 and 93 compensate the charge left on Asp140 when Asp142 rotates towards Glu144 upon substrate binding ([6], Fig. 1). We noted similar structural adjustments in the recently published structure of the D169N mutant of Chitinase 1 from Coccidioides immitis ([11]; pdb code

Acknowledgments

This work was supported by the Norwegian Research Council grants 140497/420 and 140440/130. DvA is supported by a Wellcome Trust Career Development Research Fellowship. DRH and FR are supported by BBSRC CASE studentships (Cyclacel and Syngenta, respectively). We thank Graham Gooday and Shohei Sakuda for supplying us with allosamidin and Xiaohong Jia and Ellen Hasle Kokkim for technical assistance. We thank the European Synchrotron Facility (Grenoble) for the time at beamline ID14EH1. The

References (32)

  • I. Tews et al.

    Substrate-assisted catalysis unifies two families of chitinolytic enzymes

    J. Am. Chem. Soc.

    (1997)
  • A.C. Terwisscha van Scheltinga et al.

    Stereochemistry of chitin hydrolysis by a plant chitinase/lysozyme and X-ray structure of a complex with allosamidin: evidence for substrate assisted catalysis

    Biochemistry

    (1995)
  • D.M.F. van Aalten et al.

    Structural insights into the catalytic mechanism of a family 18 exo-chitinase

    Proc. Natl. Acad. Sci. U. S. A.

    (2001)
  • M.B. Brurberg et al.

    Comparative studies of chitinases A and B from Serratia marcescens

    Microbiology

    (1996)
  • D.M.F. van Aalten et al.

    Structure of a two-domain chitotriosidase from Serratia marcescens at 1.9-A resolution

    Proc. Natl. Acad. Sci. U. S. A.

    (2000)
  • Y. Papanikolau et al.

    High resolution structural analyses of mutant chitinase A complexes with substrates provide new insight into the mechanism of catalysis

    Biochemistry

    (2001)
  • Cited by (59)

    • Pesticidal prospectives of chitinolytic bacteria in agricultural pest management

      2018, Soil Biology and Biochemistry
      Citation Excerpt :

      They are also different in their catalytic mechanisms that family 18 enzymes operate with overall retention of anomeric configuration at the cleavage point, whereas, the family 19 operate by inversion of anomeric configuration (Henrissat, 1999). Additionally, it is noteworthy that the family 18 chitinases share conserved catalytic motif of DXDXE residues (the glutamic acid is involved in protonation of oxygen in scissile glycoside bond) (Vaaje-Kolstad et al., 2004). Whereas, the family 19 candidates show no sequence similarity but attain a common tertiary structure as that of lysozymes (Frederiksen et al., 2013).

    • Characterisation of a chitinase from Pseudoalteromonas sp. DL-6, a marine psychrophilic bacterium

      2014, International Journal of Biological Macromolecules
      Citation Excerpt :

      Two diagnostic motifs (99SxGG102 and 154DxxDxDxE161), a signature of GH18 chitinases, are conserved in ChiA, suggesting that it belongs to the GH18 family. Family 18 chitinases have catalytic domains with a triosephosphate isomerase (TIM barrel) fold, and they catalyse the hydrolytic reaction by a substrate-assisted mechanism [21–23]. Family 18 chitinases can be classified into three subfamilies, A, B, and C, based on amino acid sequence similarity [24].

    View all citing articles on Scopus
    View full text