Comparative study of the reaction mechanism of family 18 chitinases from plants and microbes

Chiye Sasaki, Ai Yokoyama, Yoshifumi Itoh, Masayuki Hashimoto, Takeshi Watanabe, Tamo Fukamizo

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

Hydrolytic mechanisms of family 18 chitinases from rice (Oryza sativa L.) and Bacillus circulans WL-12 were comparatively studied by a combination of HPLC analysis of the reaction products and theoretical calculation of reaction time-courses. All of the enzymes tested produced β-anomers from chitin hexasaccharide [(GlcNAc)6], indicating that they catalyze the hydrolysis through a retaining mechanism. The rice chitinases hydrolyzed predominantly the fourth and fifth glycosidic linkages from the nonreducing end of (GlcNAc)6, whereas B. circulans chitinase A1 hydrolyzed the second linkage from the nonreducing end. In addition, the Bacillus enzyme efficiently catalyzed transglycosylation, producing significant amounts of chitin oligomers larger than the initial substrate, but the rice chitinases did not. The time-courses of (GlcNAc)6 degradation obtained by HPLC were analyzed by theoretical calculation, and the subsite structures of the rice chitinases were identified to be (-4)(-3)(-2)(-1)(+1) (+2). From the HPLC profile of the reaction products previously reported [Terwisscha van Scheltinga et al. (1995) Biochemistry 34, 15619-15623], family 18 chitinase from rubber tree (Hevea brasiliensis) was estimated to have the same type of subsite structure. Theoretical analysis of the reaction time-course for the Bacillus enzyme revealed that the enzyme has (-2)(-1) (+1)(+2)(+3)(+4)-type subsite structure, which is identical to that of fungal chitinase from Coccidioides immitis [Fukamizo et al. (2001) Biochemistry 40, 2448-2454]. The Bacillus enzyme also resembled the fungal chitinase in its transglycosylation activity. Minor structural differences between plant and microbial enzymes appear to result in such functional variations, even though all of these chitinases are classified into the identical family of glycosyl hydrolases.

Original languageEnglish
Pages (from-to)557-564
Number of pages8
JournalJournal of biochemistry
Volume131
Issue number4
DOIs
Publication statusPublished - 2002 Jan 1

Fingerprint

Chitinases
Bacilli
Bacillus
Enzymes
Hevea
Biochemistry
Chitin
High Pressure Liquid Chromatography
Reaction products
Coccidioides
Rubber
Hydrolases
Oligomers
Hydrolysis
Oryza
Degradation

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Sasaki, Chiye ; Yokoyama, Ai ; Itoh, Yoshifumi ; Hashimoto, Masayuki ; Watanabe, Takeshi ; Fukamizo, Tamo. / Comparative study of the reaction mechanism of family 18 chitinases from plants and microbes. In: Journal of biochemistry. 2002 ; Vol. 131, No. 4. pp. 557-564.
@article{5003322b5a5046e7a9dd88d4194a01b3,
title = "Comparative study of the reaction mechanism of family 18 chitinases from plants and microbes",
abstract = "Hydrolytic mechanisms of family 18 chitinases from rice (Oryza sativa L.) and Bacillus circulans WL-12 were comparatively studied by a combination of HPLC analysis of the reaction products and theoretical calculation of reaction time-courses. All of the enzymes tested produced β-anomers from chitin hexasaccharide [(GlcNAc)6], indicating that they catalyze the hydrolysis through a retaining mechanism. The rice chitinases hydrolyzed predominantly the fourth and fifth glycosidic linkages from the nonreducing end of (GlcNAc)6, whereas B. circulans chitinase A1 hydrolyzed the second linkage from the nonreducing end. In addition, the Bacillus enzyme efficiently catalyzed transglycosylation, producing significant amounts of chitin oligomers larger than the initial substrate, but the rice chitinases did not. The time-courses of (GlcNAc)6 degradation obtained by HPLC were analyzed by theoretical calculation, and the subsite structures of the rice chitinases were identified to be (-4)(-3)(-2)(-1)(+1) (+2). From the HPLC profile of the reaction products previously reported [Terwisscha van Scheltinga et al. (1995) Biochemistry 34, 15619-15623], family 18 chitinase from rubber tree (Hevea brasiliensis) was estimated to have the same type of subsite structure. Theoretical analysis of the reaction time-course for the Bacillus enzyme revealed that the enzyme has (-2)(-1) (+1)(+2)(+3)(+4)-type subsite structure, which is identical to that of fungal chitinase from Coccidioides immitis [Fukamizo et al. (2001) Biochemistry 40, 2448-2454]. The Bacillus enzyme also resembled the fungal chitinase in its transglycosylation activity. Minor structural differences between plant and microbial enzymes appear to result in such functional variations, even though all of these chitinases are classified into the identical family of glycosyl hydrolases.",
author = "Chiye Sasaki and Ai Yokoyama and Yoshifumi Itoh and Masayuki Hashimoto and Takeshi Watanabe and Tamo Fukamizo",
year = "2002",
month = "1",
day = "1",
doi = "10.1093/oxfordjournals.jbchem.a003134",
language = "English",
volume = "131",
pages = "557--564",
journal = "Journal of Biochemistry",
issn = "0021-924X",
publisher = "Oxford University Press",
number = "4",

}

Comparative study of the reaction mechanism of family 18 chitinases from plants and microbes. / Sasaki, Chiye; Yokoyama, Ai; Itoh, Yoshifumi; Hashimoto, Masayuki; Watanabe, Takeshi; Fukamizo, Tamo.

In: Journal of biochemistry, Vol. 131, No. 4, 01.01.2002, p. 557-564.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Comparative study of the reaction mechanism of family 18 chitinases from plants and microbes

AU - Sasaki, Chiye

AU - Yokoyama, Ai

AU - Itoh, Yoshifumi

AU - Hashimoto, Masayuki

AU - Watanabe, Takeshi

AU - Fukamizo, Tamo

PY - 2002/1/1

Y1 - 2002/1/1

N2 - Hydrolytic mechanisms of family 18 chitinases from rice (Oryza sativa L.) and Bacillus circulans WL-12 were comparatively studied by a combination of HPLC analysis of the reaction products and theoretical calculation of reaction time-courses. All of the enzymes tested produced β-anomers from chitin hexasaccharide [(GlcNAc)6], indicating that they catalyze the hydrolysis through a retaining mechanism. The rice chitinases hydrolyzed predominantly the fourth and fifth glycosidic linkages from the nonreducing end of (GlcNAc)6, whereas B. circulans chitinase A1 hydrolyzed the second linkage from the nonreducing end. In addition, the Bacillus enzyme efficiently catalyzed transglycosylation, producing significant amounts of chitin oligomers larger than the initial substrate, but the rice chitinases did not. The time-courses of (GlcNAc)6 degradation obtained by HPLC were analyzed by theoretical calculation, and the subsite structures of the rice chitinases were identified to be (-4)(-3)(-2)(-1)(+1) (+2). From the HPLC profile of the reaction products previously reported [Terwisscha van Scheltinga et al. (1995) Biochemistry 34, 15619-15623], family 18 chitinase from rubber tree (Hevea brasiliensis) was estimated to have the same type of subsite structure. Theoretical analysis of the reaction time-course for the Bacillus enzyme revealed that the enzyme has (-2)(-1) (+1)(+2)(+3)(+4)-type subsite structure, which is identical to that of fungal chitinase from Coccidioides immitis [Fukamizo et al. (2001) Biochemistry 40, 2448-2454]. The Bacillus enzyme also resembled the fungal chitinase in its transglycosylation activity. Minor structural differences between plant and microbial enzymes appear to result in such functional variations, even though all of these chitinases are classified into the identical family of glycosyl hydrolases.

AB - Hydrolytic mechanisms of family 18 chitinases from rice (Oryza sativa L.) and Bacillus circulans WL-12 were comparatively studied by a combination of HPLC analysis of the reaction products and theoretical calculation of reaction time-courses. All of the enzymes tested produced β-anomers from chitin hexasaccharide [(GlcNAc)6], indicating that they catalyze the hydrolysis through a retaining mechanism. The rice chitinases hydrolyzed predominantly the fourth and fifth glycosidic linkages from the nonreducing end of (GlcNAc)6, whereas B. circulans chitinase A1 hydrolyzed the second linkage from the nonreducing end. In addition, the Bacillus enzyme efficiently catalyzed transglycosylation, producing significant amounts of chitin oligomers larger than the initial substrate, but the rice chitinases did not. The time-courses of (GlcNAc)6 degradation obtained by HPLC were analyzed by theoretical calculation, and the subsite structures of the rice chitinases were identified to be (-4)(-3)(-2)(-1)(+1) (+2). From the HPLC profile of the reaction products previously reported [Terwisscha van Scheltinga et al. (1995) Biochemistry 34, 15619-15623], family 18 chitinase from rubber tree (Hevea brasiliensis) was estimated to have the same type of subsite structure. Theoretical analysis of the reaction time-course for the Bacillus enzyme revealed that the enzyme has (-2)(-1) (+1)(+2)(+3)(+4)-type subsite structure, which is identical to that of fungal chitinase from Coccidioides immitis [Fukamizo et al. (2001) Biochemistry 40, 2448-2454]. The Bacillus enzyme also resembled the fungal chitinase in its transglycosylation activity. Minor structural differences between plant and microbial enzymes appear to result in such functional variations, even though all of these chitinases are classified into the identical family of glycosyl hydrolases.

UR - http://www.scopus.com/inward/record.url?scp=0036232796&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036232796&partnerID=8YFLogxK

U2 - 10.1093/oxfordjournals.jbchem.a003134

DO - 10.1093/oxfordjournals.jbchem.a003134

M3 - Article

VL - 131

SP - 557

EP - 564

JO - Journal of Biochemistry

JF - Journal of Biochemistry

SN - 0021-924X

IS - 4

ER -