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. 1994 Aug;176(15):4465–4472. doi: 10.1128/jb.176.15.4465-4472.1994

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

T Watanabe 1, Y Ito 1, T Yamada 1, M Hashimoto 1, S Sekine 1, H Tanaka 1
PMCID: PMC196264  PMID: 8045877

Abstract

The mature form of chitinase A1 from Bacillus circulans WL-12 comprises a C-terminal domain, two type III modules (domains), and a large N-terminal domain which contains the catalytic site of the enzyme. In order to better define the roles of these chitinase domains in chitin degradation, modified chiA genes encoding various deletions of chitinase A1 were constructed. The modified chiA genes were expressed in Escherichia coli, and the gene products were analyzed after purification by high-performance liquid chromatography. Intact chitinase A1 specifically bound to chitin, while it did not show significant binding activity towards partially acetylated chitosan and other insoluble polysaccharides. Chitinases lacking the C-terminal domain lost much of this binding activity to chitin as well as colloidal chitin-hydrolyzing activity. Deletion of the type III domains, on the other hand, did not affect chitin-binding activity but did result in significantly decreased colloidal chitin-hydrolyzing activity. Hydrolysis of low-molecular-weight substrates, soluble high-molecular-weight substrates, and insoluble high-molecular-weight substrates to which chitinase A1 does not bind were not significantly affected by these deletions. Thus, it was concluded that the C-terminal domain is a chitin-binding domain required for the specific binding to chitin and that this chitin-binding activity is important for efficient hydrolysis of the sufficiently acetylated chitin. Type III modules are not directly involved in the chitin binding but play an important functional role in the hydrolysis of chitin by the enzyme bound to chitin.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ames G. F. Resolution of bacterial proteins by polyacrylamide gel electrophoresis on slabs. Membrane, soluble, and periplasmic fractions. J Biol Chem. 1974 Jan 25;249(2):634–644. [PubMed] [Google Scholar]
  2. Blaak H., Schnellmann J., Walter S., Henrissat B., Schrempf H. Characteristics of an exochitinase from Streptomyces olivaceoviridis, its corresponding gene, putative protein domains and relationship to other chitinases. Eur J Biochem. 1993 Jun 15;214(3):659–669. doi: 10.1111/j.1432-1033.1993.tb17966.x. [DOI] [PubMed] [Google Scholar]
  3. Bork P., Doolittle R. F. Proposed acquisition of an animal protein domain by bacteria. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8990–8994. doi: 10.1073/pnas.89.19.8990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fujii T., Miyashita K. Multiple domain structure in a chitinase gene (chiC) of Streptomyces lividans. J Gen Microbiol. 1993 Apr;139(4):677–686. doi: 10.1099/00221287-139-4-677. [DOI] [PubMed] [Google Scholar]
  5. Gilkes N. R., Warren R. A., Miller R. C., Jr, Kilburn D. G. Precise excision of the cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis. J Biol Chem. 1988 Jul 25;263(21):10401–10407. [PubMed] [Google Scholar]
  6. Hansen C. K. Fibronectin type III-like sequences and a new domain type in prokaryotic depolymerases with insoluble substrates. FEBS Lett. 1992 Jun 29;305(2):91–96. doi: 10.1016/0014-5793(92)80871-d. [DOI] [PubMed] [Google Scholar]
  7. Iseli B., Boller T., Neuhaus J. M. The N-terminal cysteine-rich domain of tobacco class I chitinase is essential for chitin binding but not for catalytic or antifungal activity. Plant Physiol. 1993 Sep;103(1):221–226. doi: 10.1104/pp.103.1.221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jones J. D., Grady K. L., Suslow T. V., Bedbrook J. R. Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens. EMBO J. 1986 Mar;5(3):467–473. doi: 10.1002/j.1460-2075.1986.tb04235.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kuranda M. J., Robbins P. W. Chitinase is required for cell separation during growth of Saccharomyces cerevisiae. J Biol Chem. 1991 Oct 15;266(29):19758–19767. [PubMed] [Google Scholar]
  11. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Manoil C., Beckwith J. A genetic approach to analyzing membrane protein topology. Science. 1986 Sep 26;233(4771):1403–1408. doi: 10.1126/science.3529391. [DOI] [PubMed] [Google Scholar]
  14. Meinke A., Gilkes N. R., Kilburn D. G., Miller R. C., Jr, Warren R. A. Multiple domains in endoglucanase B (CenB) from Cellulomonas fimi: functions and relatedness to domains in other polypeptides. J Bacteriol. 1991 Nov;173(22):7126–7135. doi: 10.1128/jb.173.22.7126-7135.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Molano J., Durán A., Cabib E. A rapid and sensitive assay for chitinase using tritiated chitin. Anal Biochem. 1977 Dec;83(2):648–656. doi: 10.1016/0003-2697(77)90069-0. [DOI] [PubMed] [Google Scholar]
  16. O'Neill G., Goh S. H., Warren R. A., Kilburn D. G., Miller R. C., Jr Structure of the gene encoding the exoglucanase of Cellulomonas fimi. Gene. 1986;44(2-3):325–330. doi: 10.1016/0378-1119(86)90197-6. [DOI] [PubMed] [Google Scholar]
  17. Robbins P. W., Overbye K., Albright C., Benfield B., Pero J. Cloning and high-level expression of chitinase-encoding gene of Streptomyces plicatus. Gene. 1992 Feb 1;111(1):69–76. doi: 10.1016/0378-1119(92)90604-n. [DOI] [PubMed] [Google Scholar]
  18. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tomme P., Van Tilbeurgh H., Pettersson G., Van Damme J., Vandekerckhove J., Knowles J., Teeri T., Claeyssens M. Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis. Eur J Biochem. 1988 Jan 4;170(3):575–581. doi: 10.1111/j.1432-1033.1988.tb13736.x. [DOI] [PubMed] [Google Scholar]
  20. Tsujibo H., Orikoshi H., Tanno H., Fujimoto K., Miyamoto K., Imada C., Okami Y., Inamori Y. Cloning, sequence, and expression of a chitinase gene from a marine bacterium, Altermonas sp. strain O-7. J Bacteriol. 1993 Jan;175(1):176–181. doi: 10.1128/jb.175.1.176-181.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Watanabe T., Kasahara N., Aida K., Tanaka H. Three N-terminal domains of beta-1,3-glucanase A1 are involved in binding to insoluble beta-1,3-glucan. J Bacteriol. 1992 Jan;174(1):186–190. doi: 10.1128/jb.174.1.186-190.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Watanabe T., Kobori K., Miyashita K., Fujii T., Sakai H., Uchida M., Tanaka H. Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity. J Biol Chem. 1993 Sep 5;268(25):18567–18572. [PubMed] [Google Scholar]
  23. Watanabe T., Oyanagi W., Suzuki K., Ohnishi K., Tanaka H. Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enzyme to other prokaryotic chitinases and class III plant chitinases. J Bacteriol. 1992 Jan;174(2):408–414. doi: 10.1128/jb.174.2.408-414.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Watanabe T., Oyanagi W., Suzuki K., Tanaka H. Chitinase system of Bacillus circulans WL-12 and importance of chitinase A1 in chitin degradation. J Bacteriol. 1990 Jul;172(7):4017–4022. doi: 10.1128/jb.172.7.4017-4022.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Watanabe T., Suzuki K., Oyanagi W., Ohnishi K., Tanaka H. Gene cloning of chitinase A1 from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type III homology units of fibronectin. J Biol Chem. 1990 Sep 15;265(26):15659–15665. [PubMed] [Google Scholar]

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