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

Takeshi Tanaka,^1,2, Fumikazu Takahashi,¹ Toshiaki Fukui,^1, Shinsuke Fujiwara,² Haruyuki Atomi,¹ and Tadayuki Imanaka^1*

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510,¹ Department of Bioscience, Nanobiotechnology Research Center, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337, Japan²

Received 7 May 2005/ Accepted 27 July 2005

A key step in amino sugar metabolism is the interconversion between fructose-6-phosphate (Fru6P) and glucosamine-6-phosphate (GlcN6P). This conversion is catalyzed in the catabolic and anabolic directions by GlcN6P deaminase and GlcN6P synthase, respectively, two enzymes that show no relationship with one another in terms of primary structure. In this study, we examined the catalytic properties and regulatory features of the glmD gene product (GlmD_Tk) present within a chitin degradation gene cluster in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Although the protein GlmD_Tk was predicted as a probable sugar isomerase related to the C-terminal sugar isomerase domain of GlcN6P synthase, the recombinant GlmD_Tk clearly exhibited GlcN6P deaminase activity, generating Fru6P and ammonia from GlcN6P. This enzyme also catalyzed the reverse reaction, the ammonia-dependent amination/isomerization of Fru6P to GlcN6P, whereas no GlcN6P synthase activity dependent on glutamine was observed. Kinetic analyses clarified the preference of this enzyme for the deaminase reaction rather than the reverse one, consistent with the catabolic function of GlmD_Tk. In T. kodakaraensis cells, glmD_Tk was polycistronically transcribed together with upstream genes encoding an ABC transporter and a downstream exo-ß-glucosaminidase gene (glmA_Tk) within the gene cluster, and their expression was induced by the chitin degradation intermediate, diacetylchitobiose. The results presented here indicate that GlmD_Tk is actually a GlcN6P deaminase functioning in the entry of chitin-derived monosaccharides to glycolysis in this hyperthermophile. This enzyme is the first example of an archaeal GlcN6P deaminase and is a structurally novel type distinct from any previously known GlcN6P deaminase.

Present address: Nanotechnology Research Institute, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan.

Present address: Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.

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