Sequence and Structural Analysis of the Chitinase Insertion Domain Reveals Two Conserved Motifs Involved in Chitin-Binding

Background Chitinases are prevalent in life and are found in species including archaea, bacteria, fungi, plants, and animals. They break down chitin, which is the second most abundant carbohydrate in nature after cellulose. Hence, they are important for maintaining a balance between carbon and nitrogen trapped as insoluble chitin in biomass. Chitinases are classified into two families, 18 and 19 glycoside hydrolases. In addition to a catalytic domain, which is a triosephosphate isomerase barrel, many family 18 chitinases contain another module, i.e., chitinase insertion domain. While numerous studies focus on the biological role of the catalytic domain in chitinase activity, the function of the chitinase insertion domain is not completely understood. Bioinformatics offers an important avenue in which to facilitate understanding the role of residues within the chitinase insertion domain in chitinase function. Results Twenty-seven chitinase insertion domain sequences, which include four experimentally determined structures and span five kingdoms, were aligned and analyzed using a modified sequence entropy parameter. Thirty-two positions with conserved residues were identified. The role of these conserved residues was explored by conducting a structural analysis of a number of holo-enzymes. Hydrogen bonding and van der Waals calculations revealed a distinct subset of four conserved residues constituting two sequence motifs that interact with oligosaccharides. The other conserved residues may be key to the structure, folding, and stability of this domain. Conclusions Sequence and structural studies of the chitinase insertion domains conducted within the framework of evolution identified four conserved residues which clearly interact with the substrates. Furthermore, evolutionary studies propose a link between the appearance of the chitinase insertion domain and the function of family 18 chitinases in the subfamily A.

[1]  C. Tanford,et al.  The solubility of amino acids and two glycine peptides in aqueous ethanol and dioxane solutions. Establishment of a hydrophobicity scale. , 1971, The Journal of biological chemistry.

[2]  N. Saitou,et al.  The neighbor-joining method: a new method for reconstructing phylogenetic trees. , 1987, Molecular biology and evolution.

[3]  G. Gooday.  The Ecology of Chitin Degradation , 1990 .

[4]  C. Sander,et al.  Database of homology‐derived protein structures and the structural meaning of sequence alignment , 1991, Proteins.

[5]  P. Robbins,et al.  Chitinase is required for cell separation during growth of Saccharomyces cerevisiae. , 1991, The Journal of biological chemistry.

[6]  T. Watanabe,et al.  Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity. , 1993, The Journal of biological chemistry.

[7]  K. H. Kalk,et al.  Crystal structures of hevamine, a plant defence protein with chitinase and lysozyme activity, and its complex with an inhibitor. , 1994, Structure.

[8]  M. Sela-Buurlage,et al.  A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity. , 1994, The Plant journal : for cell and molecular biology.

[9]  V. Eijsink,et al.  Characterization of a chitinase gene (chiA) from Serratia marcescens BJL200 and one-step purification of the gene product. , 1994, FEMS microbiology letters.

[10]  K S Wilson,et al.  Crystal structure of a bacterial chitinase at 2.3 A resolution. , 1994, Structure.

[11]  P. Roey,et al.  Crystal structure of endo-beta-N-acetylglucosaminidase H at 1.9 A resolution: active-site geometry and substrate recognition. , 1995, Structure.

[12]  A G Murzin,et al.  SCOP: a structural classification of proteins database for the investigation of sequences and structures. , 1995, Journal of molecular biology.

[13]  M. Hennig,et al.  Crystal structure of concanavalin B at 1.65 A resolution. An "inactivated" chitinase from seeds of Canavalia ensiformis. , 1995, Journal of molecular biology.

[14]  M. Hennig,et al.  The 1.8 A resolution structure of hevamine, a plant chitinase/lysozyme, and analysis of the conserved sequence and structure motifs of glycosyl hydrolase family 18. , 1996, Journal of Molecular Biology.

[15]  W. Goddard,et al.  The role of enzyme distortion in the single displacement mechanism of family 19 chitinases. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[16]  A. Muijsers,et al.  Chitotriosidase, a chitinase, and the 39-kDa human cartilage glycoprotein, a chitin-binding lectin, are homologues of family 18 glycosyl hydrolases secreted by human macrophages. , 1998, European journal of biochemistry.

[17]  P. Roey,et al.  Mutations of endo‐β‐N‐acetylglucosaminidase H active site residues Asp 130 and Glu 132: Activities and conformations , 2008, Protein science : a publication of the Protein Society.

[18]  C. Orengo,et al.  Evolution of protein function, from a structural perspective. , 1999, Current opinion in chemical biology.

[19]  B. Henrissat,et al.  The third chitinase gene (chiC) of Serratia marcescens 2170 and the relationship of its product to other bacterial chitinases. , 1999, The Biochemical journal.

[20]  B. Synstad,et al.  Structure of a two-domain chitotriosidase from Serratia marcescens at 1.9-A resolution. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[21]  R. Cox,et al.  The X‐ray structure of a chitinase from the pathogenic fungus Coccidioides immitis , 2008, Protein science : a publication of the Protein Society.

[22]  A. Tarentino,et al.  Structural basis for the substrate specificity of endo-beta-N-acetylglucosaminidase F(3). , 2000, Biochemistry.

[23]  Liam J. McGuffin,et al.  The PSIPRED protein structure prediction server , 2000, Bioinform..

[24]  A C Chang,et al.  The Crystal Structure of a Novel Mammalian Lectin, Ym1, Suggests a Saccharide Binding Site* , 2001, The Journal of Biological Chemistry.

[25]  J. Thornton,et al.  The (βα)8 glycosidases: sequence and structure analyses suggest distant evolutionary relationships , 2001 .

[26]  V. Subramaniam,et al.  Aromatic Amino Acids Are Critical for Stability of the Bicoid Homeodomain* , 2001, The Journal of Biological Chemistry.

[27]  S. Gåseidnes,et al.  Structural insights into the catalytic mechanism of a family 18 exo-chitinase , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Philip E. Bourne,et al.  A New Algorithm for the Alignment of Multiple Protein Structures Using Monte Carlo Optimization , 2000, Pacific Symposium on Biocomputing.

[29]  A. Oppenheim,et al.  High resolution structural analyses of mutant chitinase A complexes with substrates provide new insight into the mechanism of catalysis. , 2001, Biochemistry.

[30]  Annabel E. Todd,et al.  Evolution of function in protein superfamilies, from a structural perspective. , 2001, Journal of molecular biology.

[31]  T. Uchiyama,et al.  Roles of the Exposed Aromatic Residues in Crystalline Chitin Hydrolysis by Chitinase A from Serratia marcescens2170* , 2001, The Journal of Biological Chemistry.

[32]  D. V. van Aalten,et al.  Structure of Human Chitotriosidase , 2002, The Journal of Biological Chemistry.

[33]  C. Orengo,et al.  One fold with many functions: the evolutionary relationships between TIM barrel families based on their sequences, structures and functions. , 2002, Journal of molecular biology.

[34]  T. Uchiyama,et al.  Chitinases A, B, and C1 of Serratia marcescens 2170 Produced by Recombinant Escherichia coli: Enzymatic Properties and Synergism on Chitin Degradation , 2002, Bioscience, biotechnology, and biochemistry.

[35]  T. Taira,et al.  Antifungal Activity of Rye (Secale cereale) Seed Chitinases: the Different Binding Manner of Class I and Class II Chitinases to the Fungal Cell Walls , 2002, Bioscience, biotechnology, and biochemistry.

[36]  A. Monzingo,et al.  The structure of an allosamidin complex with the Coccidioides immitis chitinase defines a role for a second acid residue in substrate-assisted mechanism. , 2002, Journal of molecular biology.

[37]  A. Llera,et al.  Crystal Structure of Imaginal Disc Growth Factor-2 , 2002, The Journal of Biological Chemistry.

[38]  J. Madura,et al.  Family 18 chitinase-oligosaccharide substrate interaction: subsite preference and anomer selectivity of Serratia marcescens chitinase A. , 2003, The Biochemical journal.

[39]  Keith Brew,et al.  Conserved signature proposed for folding in the lipocalin superfamily , 2003, FEBS letters.

[40]  Structure of human chitotriosidase , 2003 .

[41]  Z. Luthey-Schulten,et al.  From Sequence to Structure , 2003 .

[42]  M. Hashimoto,et al.  Aromatic residues within the substrate-binding cleft of Bacillus circulans chitinase A1 are essential for hydrolysis of crystalline chitin. , 2003, The Biochemical journal.

[43]  Ming-Qun Xu,et al.  A single surface tryptophan in the chitin-binding domain from Bacillus circulans chitinase A1 plays a pivotal role in binding chitin and can be modified to create an elutable affinity tag. , 2003, Biochimica et biophysica acta.

[44]  O. Schueler‐Furman,et al.  Conserved residue clustering and protein structure prediction , 2003, Proteins.

[45]  K. H. Kalk,et al.  Crystal Structure and Carbohydrate-binding Properties of the Human Cartilage Glycoprotein-39* , 2003, Journal of Biological Chemistry.

[46]  J M Ramalho-Ortigão,et al.  Molecular characterization of Llchit1, a midgut chitinase cDNA from the leishmaniasis vector Lutzomyia longipalpis. , 2003, Insect biochemistry and molecular biology.

[47]  D. V. van Aalten,et al.  Crystal Structures of Allosamidin Derivatives in Complex with Human Macrophage Chitinase* , 2003, Journal of Biological Chemistry.

[48]  H. Merzendorfer,et al.  Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases , 2003, Journal of Experimental Biology.

[49]  Alain Roussel,et al.  Structural analysis of xylanase inhibitor protein I (XIP-I), a proteinaceous xylanase inhibitor from wheat (Triticum aestivum, var. Soisson). , 2003, The Biochemical journal.

[50]  T. Uchiyama,et al.  Importance of exposed aromatic residues in chitinase B from Serratia marcescens 2170 for crystalline chitin hydrolysis. , 2004, Journal of biochemistry.

[51]  B. Synstad,et al.  Interactions of a Family 18 Chitinase with the Designed Inhibitor HM508 and Its Degradation Product, Chitobiono-δ-lactone* , 2004, Journal of Biological Chemistry.

[52]  R. Duncan,et al.  An endochitinase A from Vibrio carchariae: cloning, expression, mass and sequence analyses, and chitin hydrolysis. , 2004, Archives of biochemistry and biophysics.

[53]  Geoffrey J. Barton,et al.  The Jalview Java alignment editor , 2004, Bioinform..

[54]  Ryo Kanai,et al.  Distribution and Phylogenetic Analysis of Family 19 Chitinases in Actinobacteria , 2004, Applied and Environmental Microbiology.

[55]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[56]  T. Shimada,et al.  The BmChi-h gene, a bacterial-type chitinase gene of Bombyx mori, encodes a functional exochitinase that plays a role in the chitin degradation during the molting process. , 2005, Insect biochemistry and molecular biology.

[57]  C. Orengo,et al.  Protein Superfamily Evolution and the Last Universal Common Ancestor (LUCA) , 2006, Journal of Molecular Evolution.

[58]  T. Fukui,et al.  Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes. , 2005, Genome research.

[59]  A. Verhoeven,et al.  Characterization of human phagocyte-derived chitotriosidase, a component of innate immunity. , 2005, International immunology.

[60]  Duochuan Li,et al.  Review of Fungal Chitinases , 2006, Mycopathologia.

[61]  Sujata Sharma,et al.  Crystal structure of a secretory signalling glycoprotein from sheep at 2.0A resolution. , 2006, Journal of structural biology.

[62]  B. Dalhus,et al.  Crystal structure and enzymatic properties of a bacterial family 19 chitinase reveal differences from plant enzymes , 2006, The FEBS journal.

[63]  P. Sikorski,et al.  Serratia marcescens chitinases with tunnel-shaped substrate-binding grooves show endo activity and different degrees of processivity during enzymatic hydrolysis of chitosan. , 2006, Biochemistry.

[64]  G. V. Goersch,et al.  cDNA cloning and 1.75 Å crystal structure determination of PPL2, an endochitinase and N‐acetylglucosamine‐binding hemagglutinin from Parkia platycephala seeds , 2006, The FEBS journal.

[65]  Rupinder Tewari,et al.  Biotechnological aspects of chitinolytic enzymes: a review , 2006, Applied Microbiology and Biotechnology.

[66]  B. Synstad,et al.  Endo/exo mechanism and processivity of family 18 chitinases produced by Serratia marcescens , 2006, The FEBS journal.

[67]  Rodrigo Lopez,et al.  Clustal W and Clustal X version 2.0 , 2007, Bioinform..

[68]  D. Speijer,et al.  Evolution of Mammalian Chitinase(-Like) Members of Family 18 Glycosyl Hydrolases , 2007, Genetics.

[69]  K. Ishikawa,et al.  Structure of the catalytic domain of the hyperthermophilic chitinase from Pyrococcus furiosus. , 2007, Acta crystallographica. Section F, Structural biology and crystallization communications.

[70]  J. Funkhouser,et al.  Chitinase family GH18: evolutionary insights from the genomic history of a diverse protein family , 2007, BMC evolutionary biology.

[71]  D. V. van Aalten,et al.  Structure of Saccharomyces cerevisiae chitinase 1 and screening-based discovery of potent inhibitors. , 2007, Chemistry & biology.

[72]  R. Robinson,et al.  Crystal structures of Vibrio harveyi chitinase A complexed with chitooligosaccharides: implications for the catalytic mechanism. , 2008, Journal of structural biology.

[73]  Joel Dudley,et al.  MEGA: A biologist-centric software for evolutionary analysis of DNA and protein sequences , 2008, Briefings Bioinform..

[74]  Nicholas Y. Palermo,et al.  The Role of Aromatic Residues in Stabilizing the Secondary and Tertiary Structure of Avian Pancreatic Polypeptide. , 2007, International journal of quantum chemistry.

[75]  C. Vorgias,et al.  Insights into the role of the (alpha+beta) insertion in the TIM-barrel catalytic domain, regarding the stability and the enzymatic activity of chitinase A from Serratia marcescens. , 2009, Biochimica et biophysica acta.

[76]  Kevin Karplus,et al.  SAM-T08, HMM-based protein structure prediction , 2009, Nucleic Acids Res..

[77]  Brandi L. Cantarel,et al.  The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics , 2008, Nucleic Acids Res..