Characterization of an exo-beta-D-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.
We previously clarified that the chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 produces diacetylchitobiose (GlcNAc(2)) as an end product from chitin. Here we sought to identify enzymes in T. kodakaraensis that were involved in the further degradation of GlcNAc(2). Through a search of the T. kodakaraensis genome, one candidate gene identified as a putative beta-glycosyl hydrolase was found in the near vicinity of the chitinase gene. The primary structure of the candidate protein was homologous to the beta-galactosidases in family 35 of glycosyl hydrolases at the N-terminal region, whereas the central region was homologous to beta-galactosidases in family 42. The purified protein from recombinant Escherichia coli clearly showed an exo-beta-D-glucosaminidase (GlcNase) activity but not beta-galactosidase activity. This GlcNase (GlmA(Tk)), a homodimer of 90-kDa subunits, exhibited highest activity toward reduced chitobiose at pH 6.0 and 80 degrees C and specifically cleaved the nonreducing terminal glycosidic bond of chitooligosaccharides. The GlcNase activity was also detected in T. kodakaraensis cells, and the expression of GlmA(Tk) was induced by GlcNAc(2) and chitin, strongly suggesting that GlmA(Tk) is involved in chitin catabolism in T. kodakaraensis. These results suggest that T. kodakaraensis, unlike other organisms, possesses a novel chitinolytic pathway where GlcNAc(2) from chitin is first deacetylated and successively hydrolyzed to glucosamine. This is the first report that reveals the primary structure of GlcNase not only from an archaeon but also from any organism.
Tanaka
Takeshi
T
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501, Japan.
Fukui
Toshiaki
T
Atomi
Haruyuki
H
Imanaka
Tadayuki
T
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002686:Chitin / Q000378:metabolism*; D006596:Hexosaminidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D002686:Chitin; D006596:Hexosaminidases; C064499:exo-beta-D-glucosaminidase