Reaction mechanism of chitosanase from <i>Streptomyces</i> sp. N174

Fukamizo, Tamo; Honda, Yoshiyuki; Goto, Susumu; Boucher, Isabelle; Brzeziński, Ryszard

doi:10.1042/bj3110377

Cited by 101 publications

(86 citation statements)

References 25 publications

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“…This mechanism is supported by the fact that microbial family 18 chitinases are unable to cleave the glycosidic bonds of GlcN in partially acetylated chitosan. On the other hand, family 19 chitinases operate by an inverting mechanism (6,17) probably involving the direct attack of the sugar anomeric carbon by a nucleophilic water molecule (35). This mechanism, which has recently been shown to also prevail in a chitosanase (6), does not involve participation of the acetamido group at C-2 and, consequently, allows the hydrolysis of ␤-linked GlcNAc as well as GlcN.…”

Section: Discussionmentioning

confidence: 99%

A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037

et al. 1996

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The specificity of chitinase C-1 of Streptomyces griseus HUT 6037 for the hydrolysis of the ␤-1,4-glycosidic linkages in partially acetylated chitosan is different from that of other microbial chitinases. In order to study the primary structure of this unique chitinase, the chiC gene specifying chitinase C-1 was cloned and its nucleotide sequence was determined. The gene encodes a polypeptide of 294 amino acids with a calculated size of 31.4 kDa. Comparison of the amino acid sequence of the deduced polypeptide with that of other proteins revealed a C-terminal catalytic domain displaying considerable sequence similarity to the catalytic domain of plant class I, II, and IV chitinases which form glycosyl hydrolase family 19. The N-terminal domain of the deduced polypeptide exhibits sequence similarity to substrate-binding domains of several microbial chitinases and cellulases but not to the chitin-binding domains of plant chitinases. The previously purified chitinase C-1 from S. griseus is suggested to be generated by proteolytic removal of the N-terminal chitin-binding domain and corresponds to the catalytic domain of the chitinase encoded by the chiC gene. High-performance liquid chromatography analysis of the hydrolysis products from N-acetyl chitotetraose revealed that chitinase C-1 catalyzes hydrolysis of the glycosidic bond with inversion of the anomeric configuration, in agreement with the previously reported inverting mechanism of plant class I chitinases. This is the first report of a family 19 chitinase found in an organism other than higher plants.Chitinases (EC 3.2.1.14) are glycosyl hydrolases which catalyze the degradation of chitin, an insoluble linear ␤-1,4-linked polymer of N-acetylglucosamine. Recently, chitinases have been receiving renewed attention because of their possible application for the biological control of chitin-containing organisms and also for the exploitation of natural chitinous materials. They are present in a wide range of organisms including bacteria, insects, viruses, plants, and animals and play important physiological and ecological roles. Chitinases so far sequenced are classified in two different families (namely, families 18 and 19) of the classification of glycosyl hydrolases based on amino acid sequence similarities (13,14). Family 18 contains chitinases from bacteria, fungi, viruses, and animals and class III and V chitinases from plants. On the other hand, class I, II, and IV plant chitinases belong to family 19, and this family solely comprises chitinases of plant origin. The two different families of chitinases display no sequence similarities with each other and have different three-dimensional structures (5).Oligosaccharides produced from partially acetylated chitosan by microbial chitinases have been previously studied in an attempt to clarify their specificity for ␤-1,4-N-acetylglucosaminic versus ␤-1,4-glucosaminic linkages (26, 27, 30). Except for chitinase C-1 from Streptomyces griseus HUT 6037, all of the examined microbial chitinases hydrolyzed GlcNAc-GlcNA...

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Section: Discussionmentioning

confidence: 99%

A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037

et al. 1996

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“…Furthermore, it was found that this chitosanase hydrolyzes (GlcN) 6 with inversion of anomeric configuration (31). The average separation between the catalytic residues, expressed as the average of the four distances measured between each pair of carboxylate oxygen atoms, depends upon the mechanism of glycosidase action (29).…”

Section: Resultsmentioning

confidence: 99%

Site-directed Mutagenesis of Evolutionary Conserved Carboxylic Amino Acids in the Chitosanase from Streptomyces sp. N174 Reveals Two Residues Essential for Catalysis

Boucher

Fukamizo

Honda

et al. 1995

Journal of Biological Chemistry

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The comparison of four sequences of prokaryotic chitosanases, belonging to the family 46 of glycosyl hydrolases, revealed a conserved N-terminal module of 50 residues, including five invariant carboxylic residues. To verify if some of these residues are important for catalytic activity in the chitosanase from Streptomyces sp. N174, these 5 residues were replaced by site-directed mutagenesis. Substitutions of Glu-22 or Asp-40 with sterically conservative (E22Q, D40N) or functionally conservative (E22D, D40E) residues reduced drastically specific activity and k cat , while K m was only slightly changed. The other residues examined, Asp-6, Glu-36, and Asp-37, retained significant activity after mutation. Circular dichroism studies of the mutant chitosanases confirmed that the observed effects are not due to changes in secondary structure. These results suggested that Glu-22 and Asp-40 are directly involved in the catalytic center of the chitosanase and the other residues are not essential for catalytic activity.

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“…chitosan. While this paper was being reviewed, the mechanism of hydrolysis of chitosan by a bacterial chitosanase was reported and indeed found to be inverting [27].…”

Section: Discussionmentioning

confidence: 99%

Plant chitinases use two different hydrolytic mechanisms

et al. 1996

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Bacterial, fungalG animal, and some plant ehNnases form fmily 18 of glycosyl hydrelases. Most plant ehitinases form the family 19, While sense ¢kitinases also have lysozyme activity, animal ~mzymes belong to different families. For 81ycesyl hydrobmes, two reaction mechanisms are possible, lendin8 to either retention or inversion of the anomerlc conflauratlon. We analyzed by HPLC the stereoehemleal ontcome of the hydrolysis catalyzed by cucumber and bean chitlnaes~ belonging to families 18 and 19, respectively. Cucumber ehltlnue used the reteJnlng mechanism as known for bacterial cldtlmmeJ and hen eag white lysozyme for which the mechanlmn hun been determined. In contrast, bean ¢ltltinase utalyzed the hydrolysis of cldtoollgosaeehafldes with overall inversion of anomerle configuration.Key words: Chitinase; Lysozyme; Catalytic mechanism; Anomeri¢ configuration I, Introduction Chitinases (EC 3.2.1.14) are enzymes that catalyze the hydrolysis of 13-1,4 linkages in chitin, a homopolymer of N-acetyl-~glucosamine [1]. The substrates of chitinases and lysozymes are very similar. Indeed, some chitinases also hydrolyse bacterial cell walls [1,2].Plant chitinases are a structurally diverse group with respect to their physical properties, enzymatic activities and localization [3]. Some chitinases, especially in combination with [3-1,3 glucanases, inhibit fungal growth in vitro [4], substantiating the idea that these enzymes play an important role in plant defense against pathogens, Chitinases could also release elicitors from funlpd cell walls [5], Recently it was shown that root chitinases can differentially inactivate Nod-factors produced by certain P, ht=obtmn strains and therefore may determine the specificity of tl,e bacterium-host plant interaction as well as modulate the activity of Nod-factors [6]. Chitinase may also have an important function in development, as demonstrated by the ability of a chitinase to overcome a block in embryogenesis in a mutant cell line of carrot [7]. Plant chitinases have been divided into several classes based on amino acid sequence [8], In the classification system of glycosyl hydrolases, all chitinases are grouped into two families [9]. Class I, II, IV and V chitinases form the family 19, that is only known in plants. Family 18 includes all fungal, animal and bacterial chitinases as well as plant chitinases of classes III and VI.The hydrolytic action of glycosyl hydrolases can take place with either retention or inversion of the anomeric configuration. The hydrolytic mechanism of but a few chitinases has been analyzed. Two bacterial chitinases and hevamme, a plant chitinase/lysozyme displayed a retaining mechanism [10,11]. Hen egg white lysozyme (family 22) [12] is also a retaining enzyme, while T4 lysozyme (family 24) was recently found to invert the anomeric configuration [13]. Holm and Sander [14] recently proposed that a family 19 chitinase and several lysozymes descend from a common ancestor, based on a weak folding similarity and the similar function. Goose lysozyme (Ya...

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Reaction mechanism of chitosanase from Streptomyces sp. N174

Cited by 101 publications

References 25 publications

A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037

A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037

Site-directed Mutagenesis of Evolutionary Conserved Carboxylic Amino Acids in the Chitosanase from Streptomyces sp. N174 Reveals Two Residues Essential for Catalysis

Plant chitinases use two different hydrolytic mechanisms

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