Publications

• Characterization of an exo-beta-D-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.

  Tanaka T., Fukui T., Atomi H., Imanaka T.

  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). T ... >> More

  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. << Less

  J. Bacteriol. 185:5175-5181(2003) [PubMed] [EuropePMC]

• The structure of an archaeal beta-glucosaminidase provides insight into glycoside hydrolase evolution.

  Mine S., Watanabe M., Kamachi S., Abe Y., Ueda T.

  The archaeal exo-β-d-glucosaminidase (GlmA) is a dimeric enzyme that hydrolyzes chitosan oligosaccharides into monomer glucosamines. GlmA is a member of the glycosidase hydrolase (GH)-A superfamily-subfamily 35 and is a novel enzyme in terms of its primary structure. Here, we present the crystal s ... >> More

  The archaeal exo-β-d-glucosaminidase (GlmA) is a dimeric enzyme that hydrolyzes chitosan oligosaccharides into monomer glucosamines. GlmA is a member of the glycosidase hydrolase (GH)-A superfamily-subfamily 35 and is a novel enzyme in terms of its primary structure. Here, we present the crystal structure of GlmA in complex with glucosamine at 1.27 Å resolution. The structure reveals that a monomeric form of GlmA shares structural homology with GH42 β-galactosidases, whereas most of the spatial positions of the active site residues are identical to those of GH35 β-galactosidases. We found that upon dimerization, the active site of GlmA changes shape, enhancing its ability to hydrolyze the smaller substrate in a manner similar to that of homotrimeric GH42 β-galactosidase. However, GlmA can differentiate glucosamine from galactose based on one charged residue while using the "evolutionary heritage residue" it shares with GH35 β-galactosidase. Our study suggests that GH35 and GH42 β-galactosidases evolved by exploiting the structural features of GlmA. << Less

  J. Biol. Chem. 292:4996-5006(2017) [PubMed] [EuropePMC]

• Concerted action of diacetylchitobiose deacetylase and exo-beta-D-glucosaminidase in a novel chitinolytic pathway in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.

  Tanaka T., Fukui T., Fujiwara S., Atomi H., Imanaka T.

  The hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 possesses chitinase (Tk-ChiA) and exo-beta-D-glucosaminidase (Tk-GlmA) for chitin degradation; the former produces diacetylchitobiose (GlcNAc2) from chitin, and the latter hydrolyzes chitobiose (GlcN2) to glucosamine (GlcN). To identif ... >> More

  The hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 possesses chitinase (Tk-ChiA) and exo-beta-D-glucosaminidase (Tk-GlmA) for chitin degradation; the former produces diacetylchitobiose (GlcNAc2) from chitin, and the latter hydrolyzes chitobiose (GlcN2) to glucosamine (GlcN). To identify the enzyme that physiologically links these two activities, here we focused on the deacetylase that provides the substrate for Tk-GlmA from GlcNAc2. The deacetylase could be detected in and partially purified from T. kodakaraensis cells, and the corresponding gene (Tk-dac) was identified on the genome. The deduced amino acid sequence was classified into the LmbE protein family including N-acetylglucosaminylphosphatidylinositol de-N-acetylases and 1-D-myo-inosityl-2-acetamido-2-deoxy-alpha-D-glucopyranoside deacetylase. Recombinant Tk-Dac showed deacetylase activity toward N-acetylchitooligosaccharides (GlcNAc(2-5)), and the deacetylation site was revealed to be specific at the nonreducing GlcNAc residue. The enzyme also deacetylated GlcNAc monomer. In T. kodakaraensis cells, the transcription of Tk-dac, Tk-glmA, Tk-chiA, and the clustered genes were induced by GlcNAc2, suggesting the function of this gene cluster in chitin catabolism in vivo. These results have revealed a unique chitin catabolic pathway in T. kodakaraensis, in which GlcNAc2 produced from chitin is degraded by the concerted action of Tk-Dac and Tk-GlmA. That is, GlcNAc2 is site-specifically deacetylated to GlcN-GlcNAc by Tk-Dac and then hydrolyzed to GlcN and GlcNAc by Tk-GlmA followed by a second deacetylation step of the remaining GlcNAc by Tk-Dac to form GlcN. This is the first elucidation of an archaeal chitin catabolic pathway and defines a novel mechanism for dimer processing using a combination of deacetylation and cleavage, distinct from any previously known pathway. << Less

  J. Biol. Chem. 279:30021-30027(2004) [PubMed] [EuropePMC]

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