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Structural Relationships in the Lysozyme Superfamily: Significant Evidence for Glycoside Hydrolase Signature Motifs

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Structural Relationships in the Lysozyme Superfamily: Significant Evidence for Glycoside Hydrolase Signature Motifs Structural Relationships in the Lysozyme Superfamily: Significant Evidence for Glycoside Hydrolase Signature Motifs Alexandre Wohlko¨ nig1, Joe¨lle Huet2, Yvan Looze2, Rene´ Wintjens2,3* 1 Structural Biology Brussels and Molecular and Cellular Interactions, VIB, Brussels, Belgium, 2 Laboratoire de Chimie Ge´ne´rale, Institut de Pharmacie, Universite´ Libre de Bruxelles, Brussels, Belgium, 3 Interdisciplinary Research Institute, USR 3078 CNRS, Villeneuve d’Ascq, France Abstract Background: Chitin is a polysaccharide that forms the hard, outer shell of arthropods and the cell walls of fungi and some algae. Peptidoglycan is a polymer of sugars and amino acids constituting the cell walls of most bacteria. Enzymes that are able to hydrolyze these cell membrane polymers generally play important roles for protecting plants and animals against infection with insects and pathogens. A particular group of such glycoside hydrolase enzymes share some common features in their three-dimensional structure and in their molecular mechanism, forming the lysozyme superfamily. Results: Besides having a similar fold, all known catalytic domains of glycoside hydrolase proteins of lysozyme superfamily (families and subfamilies GH19, GH22, GH23, GH24 and GH46) share in common two structural elements: the central helix of the all-a domain, which invariably contains the catalytic glutamate residue acting as general-acid catalyst, and a b-hairpin pointed towards the substrate binding cleft. The invariant b-hairpin structure is interestingly found to display the highest amino acid conservation in aligned sequences of a given family, thereby allowing to define signature motifs for each GH family. Most of such signature motifs are found to have promising performances for searching sequence databases. Our structural analysis further indicates that the GH motifs participate in enzymatic catalysis essentially by containing the catalytic water positioning residue of inverting mechanism. Conclusions: The seven families and subfamilies of the lysozyme superfamily all have in common a b-hairpin structure which displays a family-specific sequence motif. These GH b-hairpin motifs contain potentially important residues for the catalytic activity, thereby suggesting the participation of the GH motif to catalysis and also revealing a common catalytic scheme utilized by enzymes of the lysozyme superfamily. Citation: Wohlko¨nig A, Huet J, Looze Y, Wintjens R (2010) Structural Relationships in the Lysozyme Superfamily: Significant Evidence for Glycoside Hydrolase Signature Motifs. PLoS ONE 5(11): e15388. doi:10.1371/journal.pone.0015388 Editor: Vladimir N. Uversky, Indiana University, United States of America Received August 6, 2010; Accepted August 31, 2010; Published November 9, 2010 Copyright: ß 2010 Wohlko¨nig et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was supported by the Belgian Fund for Scientific Research (FNRS-FRS) (FRFC project). RW is Research Associate at the FNRS-FRC. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction The present work was initiated by our previous observation of a highly conserved sequence motif which characterizes glycoside Due to a worldwide effort of structural genomics projects, the hydrolase family 19 chitinase [20]. We wondered whether the GH number of known three-dimensional protein structures rapidly families structurally related to GH19 also possess a similar increases [1]. It is now even frequent that structures are signature motif. The 5 studied GH families, designated as the determined prior to any knowledge of their biological function lysozyme superfamily [21–22], are plant chitinase GH19 family, [2]. The ability to predict details of protein function and their C-type lysozyme GH22 family, G-type lysozyme GH23 family, V- biological role from structure becomes thus of great importance. type lysozyme GH24 and the chitosanase GH46 family (http:// To date, several methods are available for this purpose [3–8]. www.cazy.org/) [23]. Many of them are based on the occurrence of particular clusters of Lysozymes (E.C. 3.2.1.17) and chitinases (E.C. 3.2.1.14) residues, in protein sequence or in protein 3D structure that could represent an important class of polysaccharide-hydrolyzing give a functional role to the unknown protein [9–14]. Such clusters enzymes. Chitinase enzymes catalyse the breakdown of chitin, a can be also called patterns, motifs, signatures or fingerprints, and linear polymer found in insects, crustaceans and fungi cell walls were accumulated from various protein families in freely accessible consisting of b-1-4 linked N-acetylglucosamine (GlcNAc), while databases, such as PROSITE [15], PRINTS [16], BLOCKS [17], the lysozymes hydrolyse peptidoglycans present in bacterial cell MSDmotif [18] or FunClust [19]. The signature search is also an walls which contain alternating b-1-4 linked residues of GlcNAc effective alternative for the detection of remote protein homo- and N-acetylmuramic acid [24]. The chemical similitude between logues from low-similarity sequences. the two polysaccharide substrates leads to the fact that some PLoS ONE | www.plosone.org 1 November 2010 | Volume 5 | Issue 11 | e15388 Structural Relationships in the Lysozyme Family lysozymes can hydrolyse chitin, but less efficiently than their [39]. 32 X-ray structures were selected from the protein structure natural substrate and vice versa [25–27]. Thus, some lysozymes databank (see Methods for the selection criteria). The obtained could be considered as good chitinases and reciprocally some values of normalized structural similarity score, called DaliLile Z- chitinases can cleave peptidoglycan, the natural substrate of score, ranged from 1.2 to 47.3, and the superimposition rms values lysozymes [28]. However there is no obvious amino acid sequence from 0.6 to 4.6 A˚ . The matrix of Z-score values was transformed similarity found between these two types of enzymes [22]. On the in distance metric index and a clustering tree was generated (see other hand, a different enzyme, chitosanase (E.C. 3.2.1.132), also Methods). A jackknife procedure has been applied to test the hydrolyses polymer of GlcNAc, but with specificity for a partial reliability of the resulting tree, which indicated that, except for (over 60%) or full deacetylation of chitin, named chitosan. The some internal nodes within the GH19 and GH22c clusters, all of differences in substrate specificity of these enzymes, and the nodes were stable (Fig. 1). occasionally in their catalytic mechanism, make them belong to The obtained tree (Fig. 1) shows the similarities and differences different protein families with different E.C. number [23]. All among proteins of the lysozyme superfamilies, but also indicates these proteins could be considered, to a large extent, as chitinolytic structural relationships in a given GH family: (i) lysozyme enzymes, i.e. enzymes that are able to hydrolyze derivatives of superfamily exhibits a structural continuum with the different GH chitin [29]. families roughly structurally equidistant from each other; (ii) Chitinolytic enzymes are widely distributed in the tissues and according to our structural similarity index, the range of distances body fluids of animals, plants and microorganisms and also in the between the five GH families was 63–82; (iii) the mean distance soil- and bio-spheres of the earth. Chitinases are key enzymes in between the two more distant GH families, namely GH46 and plant defence systems against fungal infection [30–31]. They are GH19 families, was 82.4, whereas this distance was 63.4 between classified on the basis of amino acid sequence in two different GH GH23 and GH24, the two closest families; (iv) although they are families, namely GH18 and GH19 [32]. Chitinases of GH18 are grouped in a same GH family, a large distance was found between encountered in all living organisms whereas those of GH19 are the two classes of both GH22 and GH24 families, i.e. the distance mainly found in plants. Proteins of these two GH families between GH22c and GH22i was 46.7 and 57.7 between GH24l and significantly differ both in their three-dimensional structures and GH24v; (v) the chitinase GH19 family showed two structurally in their enzymatic mechanisms [33–34]. distinct clusters and a mean distance between all members of 23.3 Lysozymes are widely spread throughout nature. They are used (Table 1). One cluster grouped the plant chitinases and the second by plants and higher organisms as a first defence mechanism one the bacterial chitinases, except the recent structure of Norway against bacterial invasion [35]. Since its discovery by Fleming in spruce chitinase which was curiously grouped with the bacterial 1922, lysozyme has been extensively studied. It was one of the first chitinases; this could be explained by the fact that the latter chitinase proteins to be completely sequenced [36] and one of the first is a class IV chitinase while the other plant chitinase are class I or II; enzymes for which the X-ray structure was determined [37]. (vi) although the two structures of GH46 chitosanase family are Several classes of lysozymes have been identified on the basis of bacterial proteins, the distance between them was high (46.2), in the their sequence similarities [35]. The best known ones are of the C- same order of magnitude as distance between the two classes type (chicken-type or GH22), the G-type (goose-type or GH23) GH22c and GH22i. and the V-type (viral type or GH24). Note that the topology of the clustering tree was further Chitosanases are classified in GH46 [38]. Most of these confirmed using MAMMOTH-mult server [40], another struc- enzymes are found in microorganisms and few are found in virus ture-comparison tool, which produced a very similar result.
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