Different cleavage specificities of the dual catalytic domains in chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

J Biol Chem. 2001 Sep 21;276(38):35629-35. doi: 10.1074/jbc.M105919200. Epub 2001 Jul 23.

Abstract

The chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, Tk-ChiA, has an interesting multidomain structure containing dual catalytic domains and triple chitin-binding domains. To determine the biochemical properties of each domain, we constructed deletion mutant genes corresponding to the individual catalytic domains and purified the recombinant proteins. A synergistic effect was observed when chitin was degraded in the presence of both catalytic domains, suggesting different cleavage specificity of these domains. Analyses of degradation products from N-acetyl-chitooligosaccharides and their chromogenic derivatives with thin layer chromatography indicated that the N-terminal catalytic domain mainly hydrolyzed the second glycosidic bond from the nonreducing end of the oligomers, whereas the C-terminal domain randomly hydrolyzed glycosidic bonds other than the first bond from the nonreducing end. Both catalytic domains formed diacetyl-chitobiose as a major end product and possessed transglycosylation activity. Further analysis of degradation products from colloidal chitin with high performance liquid chromatography showed that the N-terminal catalytic domain exclusively liberated diacetyl-chitobiose, whereas reactions with the C-terminal domain led to N-acetyl-chitooligosaccharides of various lengths. These results demonstrated that the N-terminal and C-terminal catalytic domains functioned as exo- and endochitinases, respectively. The biochemical results provide a physiological explanation for the presence of two catalytic domains with different specificity and suggest a cooperative function between the two on a single polypeptide in the degradation of chitin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Catalytic Domain
  • Chitinases / metabolism*
  • DNA Primers
  • Hydrolysis
  • Mutagenesis
  • Sequence Deletion
  • Substrate Specificity
  • Thermococcus / enzymology*

Substances

  • DNA Primers
  • Chitinases