Characterization of an Exo-β- d -Glucosaminidase Involved in a Novel Chitinolytic Pathway from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

Abstract

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 ␤-glycosyl hydrolase was found in the near vicinity of the chitinase gene. The primary structure of the candidate protein was homologous to the ␤-galactosidases in family 35 of glycosyl hydrolases at the N-terminal region, whereas the central region was homologous to ␤-galactosidases in family 42. The purified protein from recombinant Escherichia coli clearly showed an exo-␤-D-glucosaminidase (GlcNase) activity but not ␤-galactosidase activity. This GlcNase (GlmA Tk ), a homodimer of 90-kDa subunits, exhibited highest activity toward reduced chitobiose at pH 6.0 and 80°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.

Description of Thermococcus kodakaraensis sp. nov., a well studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1

“…The enzyme represents a novel archaeal-type FBPase and is strictly regulated at the transcriptional level responding to the carbon source (Rashid et al 2002b). The major enzymes involved in chitin assimilation by strain KOD1 have been characterized (Tanaka et al 2001(Tanaka et al , 2003, and it has been found that the expression of these enzymes is induced only in the presence of chitin and its degradation products (Tanaka et al 2003). These features indicate that strain KOD1 can assimilate various carbon sources, and has evolved strict regulatory systems, enabling it to use these substrates efficiently.…”

Characterization of a Novel Glucosamine-6-Phosphate Deaminase from a Hyperthermophilic Archaeon

“…The GlcNAc 2 is probably translocated across the cell membrane by an ABC transport system and then deacetylated by a deacetylase (Dac Tk ) with nonreducing end specificity. The partially acetylated disaccharide GlcN-GlcNAc is hydrolyzed into GlcN and GlcNAc by an exo-␤-glucosaminidase (GlmA Tk ), and the generated GlcNAc is further deacetylated to GlcN by Dac Tk (26,27), resulting in the complete conversion of chitin into GlcN monomers. The genes for these enzymes are highly clustered on the T. kodakaraensis genome, whose features have recently been reported (accession no.…”

Perspectives of Chitin Deacetylase Research

“…Well-defined COS products have been identified and characterized using novel CDAs from different sources. For instance, CDAs from C. lindemuthianum (Tokuyasu et al 1997) and Thermococcus kodakaraensis KOD1 (Tanaka et al 2004) could exclusively convert GlcNAc-GlcNAc into GlcN-GlcNAc. Besides that, CDAs from C. lindemuthianum could also be used for an enzymatic synthesis of GlcNAc-GlcN from GlcNGlcN (Tokuyasu et al 1999) and Thermococcus kodakaraensis KOD1 could also convert GlcNAc into GlcN (Tanaka et al 2004).…”

“…For instance, CDAs from C. lindemuthianum (Tokuyasu et al 1997) and Thermococcus kodakaraensis KOD1 (Tanaka et al 2004) could exclusively convert GlcNAc-GlcNAc into GlcN-GlcNAc. Besides that, CDAs from C. lindemuthianum could also be used for an enzymatic synthesis of GlcNAc-GlcN from GlcNGlcN (Tokuyasu et al 1999) and Thermococcus kodakaraensis KOD1 could also convert GlcNAc into GlcN (Tanaka et al 2004). CDA from Vibrio cholerae could exclusively convert GlcNAcGlcNAc into GlcNAc-GlcN (Li et al 2007), while CDA from M. rouxii and Absidia corulea could not deacetylate GlcNAc-GlcNAc at all (Gao et al 1995, Kafetzopoulos et al 1993.…”