The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation

Watanabe, Takeshi; Ito, Y.; Yamada, Takao; Hashimoto, Masayuki; Sekine, Sakari; Tanaka, Hironori

doi:10.1128/jb.176.15.4465-4472.1994

Cited by 268 publications

(206 citation statements)

References 30 publications

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“…For example, the related regions of chitinase A1 and D of Bacillus circulans WL-12 (Fig. 4) were experimentally demonstrated to be chitin-binding domains and essential for efficient hydrolysis of insoluble chitin (40). Therefore, although the catalytic domain of chitinase C is clearly related to family 19 plant chitinases, its N-terminal domain, which probably serves as a chitin-binding domain, appears related to microbial chitinand cellulose-binding domains and not to plant chitin-binding domains.…”

Section: Resultsmentioning

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: Resultsmentioning

confidence: 99%

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

et al. 1996

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“…Each base in bold and double underlined is that at which transcription initiates and the subscript numeral beside it indicates its base number, as found in GenBank. N with bracketed numeral indicates the number of bases to the putative ribsome binding site (italics) located upstream of a putative in-frame translation initiation codon (ATG; underlined (Techkarnjanaruk and Goodman, 1999), there was a chitinase C (ChiC) domain, involved in chitin-binding (Pfam 06483; Watanabe et al, 1994), of 128 residues, Glu 793 -Gly 980 (Table 5 and Figure 1). Also, ChiA was found to have a chitin-binding type 3 domain (ChtBD3; smart 00495) at its N-terminal end of 52 residues, Cys 31 -Cys 82 (Table 5 and Figure 1).…”

Section: Tsp Potential Promotersmentioning

confidence: 99%

Nutrient regime regulates complex transcriptional start site usage within a Pseudoalteromonas chitinase gene cluster

Delpin

Goodman

2009

The ISME Journal

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The chitinase gene cluster of the marine bacterium Pseudoalteromonas sp. S91, chiABC, which produces the major chitinases of this sp., was transcribed as an operon and from each individual gene. chiA, chiB and chiC were found to possess multiple transcriptional start points (TSPs), the use of which was determined by the nutrient regime used for S91 growth. In minimal medium containing glutamate, chiA, chiB and chiC each used 3, 1 and 1 TSP, respectively. Upon the addition of the chitin monomer N-acetylglucosamine, the number of chiA TSPs was unaffected. However, chiB used an additional 4 TSPs, and chiC used four new TSPs excluding the TSP used in glutamate only. In addition, the cluster was transcribed as an operon from TSP A1 of chiA. All TSPs were potentially associated with either a r 70 -or r 54 -dependent promoter. Under the growth conditions used, no TSPs were detected for chiB or chiC in S91CX, a chiA transposon mutant. The transcription of the S91 chiABC gene cluster produced at least four polycistronic mRNAs. In addition, the occurrence of operon transcription of chiABC, and identification of an additional 12 putative TSPs within the gene cluster, gave an indication that each gene appeared to be transcribed from more than one promoter region upstream of each in-frame translation start codon. Questions arose regarding the reason for this complexity of transcription within the gene cluster, leading to a re-evaluation of the Chi protein domains. By bioinformatic review, ChiA, ChiB and ChiC were found to potentially possess additional putative domains.

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“…Important elements of the proposed mechanism were inferred from modeling studies and structures of glycosidases that do not belong to family 18 (9,15,16). Furthermore, the model does not account for the roles of several highly conserved residues in family 18 chitinases that are known to be important for catalytic activity (17)(18)(19). Substrate binding in the Ϫ subsites has been studied in hevamine, a family 18 endochitinase with an open active site architecture (8,20).…”

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Structural insights into the catalytic mechanism of a family 18 exo-chitinase

Aalten

Komander

Synstad

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

410

493

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Chitinase B (ChiB) from Serratia marcescens is a family 18 exochitinase whose catalytic domain has a TIM-barrel fold with a tunnel-shaped active site. We have solved structures of three ChiB complexes that reveal details of substrate binding, substrateassisted catalysis, and product displacement. The structure of an inactive ChiB mutant (E144Q) complexed with a pentameric substrate (binding in subsites ؊2 to ؉3) shows closure of the ''roof'' of the active site tunnel. It also shows that the sugar in the ؊1 position is distorted to a boat conformation, thus providing structural evidence in support of a previously proposed catalytic mechanism. The structures of the active enzyme complexed to allosamidin (an analogue of a proposed reaction intermediate) and of the active enzyme soaked with pentameric substrate show events after cleavage of the glycosidic bond. The latter structure shows reopening of the roof of the active site tunnel and enzyme-assisted product displacement in the ؉1 and ؉2 sites, allowing a water molecule to approach the reaction center. Catalysis is accompanied by correlated structural changes in the core of the TIM barrel that involve conserved polar residues whose functions were hitherto unknown. These changes simultaneously contribute to stabilization of the reaction intermediate and alternation of the pKa of the catalytic acid during the catalytic cycle.C hitinases hydrolyze chitin, a linear polymer of ␤-(1,4)-linked N-acetylglucosamine (NAG), which is an abundant biopolymer. These enzymes are essential to chitin-containing organisms (fungi, insects, crustaceans) and are used by several bacteria to exploit chitin as a source of energy. Chitinase inhibitors have generated a lot of interest given their potential as insecticides (1), fungicides (2, 3), and antimalarials (4, 5). Biotechnological exploitation of chitinases, as well as design of inhibitors with sufficiently high selectivity and affinity, requires detailed knowledge of the catalytic mechanism and enzyme-substrate interactions.Most nonplant chitinases belong to glycosidase family 18 (6) and degrade chitin with retention of the stereochemistry at the anomeric carbon (7-10). The reaction is thought to be initiated by distortion of the Ϫ1 sugar ring and protonation of the glycosidic oxygen by a protonated acidic residue. The subsequent nucleophilic attack differs from classical reaction mechanisms of retaining enzymes such as lysozyme (11) and amylases (12) in that it involves the N-acetyl group of the Ϫ1 sugar, rather than a carboxylate side chain on the protein (8,9,13,14). Thus, the first step of chitinolysis results in cleavage of the sugar chain and formation of an oxazolinium ion intermediate, and hydrolysis of this ion completes the reaction (9, 15) (Fig. 1).Although the current model for the catalytic mechanism is well established, the amount of structural evidence in its support is limited (8). Important elements of the proposed mechanism were inferred from modeling studies and structures of glycosidases that do not belong to f...

show abstract

The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation

Cited by 268 publications

References 30 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

Nutrient regime regulates complex transcriptional start site usage within a Pseudoalteromonas chitinase gene cluster

Structural insights into the catalytic mechanism of a family 18 exo-chitinase

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