X-ray Structure of Papaya Chitinase Reveals the Substrate Binding Mode of Glycosyl Hydrolase Family 19 Chitinases

Abstract

The crystal structure of a chitinase from Carica papaya has been solved by the molecular replacement method and is reported to a resolution of 1.5 A. This enzyme belongs to family 19 of the glycosyl hydrolases. Crystals have been obtained in the presence of N-acetyl- d-glucosamine (GlcNAc) in the crystallization solution and two well-defined GlcNAc molecules have been identified in the catalytic cleft of the enzyme, at subsites -2 and +1. These GlcNAc moieties bind to the protein via an extensive network of interactions which also involves many hydrogen bonds mediated by water molecules, underlying their role in the catalytic mechanism. A complex of the enzyme with a tetra-GlcNAc molecule has been elaborated, using the experimental interactions observed for the bound GlcNAc saccharides. This model allows to define four major substrate interacting regions in the enzyme, comprising residues located around the catalytic Glu67 (His66 and Thr69), the short segment E89-R90 containing the second catalytic residue Glu89, the region 120-124 (residues Ser120, Trp121, Tyr123, and Asn124), and the alpha-helical segment 198-202 (residues Ile198, Asn199, Gly201, and Leu202). Water molecules from the crystal structure were introduced during the modeling procedure, allowing to pinpoint several additional residues involved in ligand binding that were not previously reported in studies of poly-GlcNAc/family 19 chitinase complexes. This work underlines the role played by water-mediated hydrogen bonding in substrate binding as well as in the catalytic mechanism of the GH family 19 chitinases. Finally, a new sequence motif for family 19 chitinases has been identified between residues Tyr111 and Tyr125.

The first crystal structures of a family 19 class IV chitinase: the enzyme from Norway spruce

“…For example, the profound effects of a mutation in His211 of the Brassica enzyme (Tang et al 2004) (equivalent to His112 of PaChi) are likely to result from problems in binding/positioning of the substrate. Tyr158 (which is shown to be the site of an inactivating mutation here) has a more structural role, being part of a newly described motif that is conserved in most family 19 sequences (Huet et al 2008).…”

“…The number and location of GlcNAc subsites seem to vary. In class I/II enzymes, subsites -2 to +2 have been described (Huet et al 2008). The bacterial enzyme, which lacks loops I, II and V, has also been demonstrated experimentally to have four subsites (Hoell et al 2006).…”

“…This was thought to arise from the wide catalytic cleft, which is better suited to the binding of large, insoluble molecules rather than of small, soluble compounds. Very recently, the structure of papaya chitinase (Huet et al 2008), crystallized in the presence of very high (molar) concentrations of GlcNAc, provided vital information about chitin binding. For example, the profound effects of a mutation in His211 of the Brassica enzyme (Tang et al 2004) (equivalent to His112 of PaChi) are likely to result from problems in binding/positioning of the substrate.…”

“…One was proposed to act as a general acid that donates a proton in the reaction, and the other as a general base that activates a nucleophilic water molecule and stabilizes a charged intermediate (Brameld and Goddard 1998;Fukamizo 2000;Hahn et al 2000). The very recent publication of the structure of class I or II chitinase from papaya bound to two GlcNAc molecules (Huet et al 2008) provided a basis for modeling the binding of four GlcNAc units in the active site, and supported the proposed reaction mechanism.…”

“…Based on sequence comparisons within GH family 19 (Tang et al 2004;Ubhayasekera et al 2007) and by analogy to the structure of the papaya chitinase bound to two molecules of GlcNAc (Huet et al 2008), the active site lies in the wide cleft between the two domains. The general acid, Glu113, is part of a triad that also includes Arg230 and Glu218 (Fig.…”