Crystal Structure of Chitosanase from Bacillus circulans MH-K1 at 1.6-Å Resolution and Its Substrate Recognition Mechanism*

Chitosanase from Bacillus circulans MH-K1 is a 29-kDa extracellular protein composed of 259 amino acids. The crystal structure of chitosanase from B. circulans MH-K1 has been determined by multiwavelength anomalous diffraction method and refined to crystallographic R = 19.2% (R free = 23.5%) for the diffraction data at 1.6-Å resolution collected by synchrotron radiation. The enzyme has two globular upper and lower domains, which generate the active site cleft for the substrate binding. The overall molecular folding is similar to chitosanase from Streptomyces sp. N174, although there is only 20% identity at the amino acid sequence level between both chitosanases. However, there are three regions in which the topology is remarkably different. In addition, the disulfide bridge between Cys50 and Cys124 joins the β1 strand and the α7 helix, which is not conserved among other chitosanases. The orientation of two backbone helices, which connect the two domains, is also different and is responsible for the differences in size and shape of the active site cleft in these two chitosanases. This structural difference in the active site cleft is the reason why the enzymes specifically recognize different substrates and catalyze different types of chitosan degradation.

[1]  R. Read.  Improved Fourier Coefficients for Maps Using Phases from Partial Structures with Errors , 1986 .

[2]  M. Mitsutomi,et al.  Action pattern of Bacillus sp. no. 7-M chitosanase on partially N-acetylated chitosan. , 1992, Bioscience, biotechnology, and biochemistry.

[3]  D. Bacon,et al.  A fast algorithm for rendering space-filling molecule pictures , 1988 .

[4]  E A Merritt,et al.  Raster3D Version 2.0. A program for photorealistic molecular graphics. , 1994, Acta crystallographica. Section D, Biological crystallography.

[5]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[6]  Yoshiyuki Amemiya,et al.  A new type of X-ray area detector utilizing laser stimulated luminescence , 1986 .

[7]  R. Brzezinski,et al.  Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function. , 1997, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[8]  A. Asselin,et al.  Diversity of chitosanase activity in cucumber , 1992 .

[9]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[10]  A Bairoch,et al.  New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. , 1993, The Biochemical journal.

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

[12]  B. C. Wang.  Resolution of phase ambiguity in macromolecular crystallography. , 1985, Methods in enzymology.

[13]  T. Fujii,et al.  PURIFICATION AND PROPERTIES OF CHITOSANASE FROM BACILLUS CIRCULANS MH-K1 , 1988 .

[14]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[15]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[16]  R. Brzezinski,et al.  Site-directed Mutagenesis of Evolutionary Conserved Carboxylic Amino Acids in the Chitosanase from Streptomyces sp. N174 Reveals Two Residues Essential for Catalysis (*) , 1995, The Journal of Biological Chemistry.

[17]  D. Shah,et al.  Antifungal proteins from plants. Purification, molecular cloning, and antifungal properties of chitinases from maize seed. , 1992, The Journal of biological chemistry.

[18]  M. Matsushima,et al.  Structural changes of active site cleft and different saccharide binding modes in human lysozyme co-crystallized with hexa-N-acetyl-chitohexaose at pH 4.0. , 1995, Journal of molecular biology.

[19]  E. Marcotte,et al.  X-ray structure of an anti-fungal chitosanase from streptomyces N174 , 1996, Nature Structural Biology.

[20]  T. Yamada,et al.  Alternative expression of a chitosanase gene produces two different proteins in cells infected with Chlorella virus CVK2. , 1997, Virology.

[21]  S. Suh,et al.  Refined structure of the chitinase from barley seeds at 2.0 a resolution. , 1996, Acta crystallographica. Section D, Biological crystallography.

[22]  Y. Kagawa,et al.  PRIMARY STRUCTURE OF CHITOSANASE PRODUCED BY BACILLUS CIRCULANS MH-K1 , 1992 .

[23]  Liisa Holm,et al.  Structural similarity of plant chitinase and lysozymes from animals and phage , 1994, FEBS letters.

[24]  C. Nairn,et al.  Identification and characterization of acidic hydrolases with chitinase and chitosanase activities from sweet orange callus tissue. , 1994, Plant and Cell Physiology.

[25]  M. Okazaki,et al.  Cloning and characterization of a chitosanase gene from the plant pathogenic fungus Fusarium solani , 1996 .

[26]  A. Monzingo,et al.  The refined crystal structure of an endochitinase from Hordeum vulgare L. seeds at 1.8 A resolution. , 1995, Journal of molecular biology.

[27]  Nobuhisa Watanabe,et al.  Weissenberg camera for macromolecules with imaging plate data collection system at the Photon Factory: Present status and future plan (invited) , 1995 .

[28]  J. Masson,et al.  A new chitosanase gene from a Nocardioides sp. is a third member of glycosyl hydrolase family 46. , 1995, Microbiology.

[29]  Y. Mikami,et al.  Purification and some properties of a chitosanase of Nocardioides sp. , 1995 .

[30]  J. Verburg,et al.  Purification and Characterization of an Antifungal Chitinase from Arabidopsis thaliana. , 1991, Plant physiology.

[31]  S. Withers,et al.  Mechanisms of enzymatic glycoside hydrolysis. , 1994, Current opinion in structural biology.

[32]  J. V. Van Etten,et al.  Analysis of 94 kb of the chlorella virus PBCV-1 330-kb genome: map positions 88 to 182. , 1996, Virology.

[33]  B. Henrissat,et al.  Structures and mechanisms of glycosyl hydrolases. , 1995, Structure.

[34]  B Henrissat,et al.  A classification of glycosyl hydrolases based on amino acid sequence similarities. , 1991, The Biochemical journal.

[35]  A. Asselin,et al.  Multiple forms of chitosanase activities , 1992 .

[36]  A. Brunger.  Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. , 1992 .

[37]  A. Bolotin,et al.  A 23911 bp region of the Bacillus subtilis genome comprising genes located upstream and downstream of the lev operon. , 1997, Microbiology.

[38]  T. Fujii,et al.  Purification and characterization of an extracellular chitosanase produced by Amycolatopsis sp. CsO-2 , 1994 .

[39]  J. Masson,et al.  Primary sequence of the chitosanase from Streptomyces sp. strain N174 and comparison with other endoglycosidases. , 1994, Gene.

[40]  R. Brzezinski,et al.  Reaction mechanism of chitosanase from Streptomyces sp. N174. , 1995, The Biochemical journal.

[41]  P. Stougaard,et al.  PR-proteins accumulating in spruce roots infected with a pathogenic Pythium sp. isolate include chitinases, chitosanases and β-1,3-glucanases , 1993 .

[42]  A Bairoch,et al.  Updating the sequence-based classification of glycosyl hydrolases. , 1996, The Biochemical journal.

[43]  P. Main,et al.  The use of Sayre's equation with solvent flattening and histogram matching for phase extension and refinement of protein structures , 1990 .

[44]  P. Hart,et al.  Crystal structure of an endochitinase from Hordeum vulgare L. seeds. , 1993, Journal of molecular biology.

[45]  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.