JOURNAL OF BACTERIOLOGY, Oct. 2005, p. 7038–7044
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 187, No. 20
Characterization of a Novel Glucosamine-6-Phosphate Deaminase
from a Hyperthermophilic Archaeon
Takeshi Tanaka,1,2† Fumikazu Takahashi,1Toshiaki Fukui,1‡ Shinsuke Fujiwara,2Haruyuki Atomi,1
and Tadayuki Imanaka1*
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura,
Nishikyo-ku, Kyoto 615-8510,1and Department of Bioscience, Nanobiotechnology Research Center, School of
Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337,2Japan
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 (GlmDTk) present within a chitin degradation gene cluster in the hyperthermophilic
archaeon Thermococcus kodakaraensis KOD1. Although the protein GlmDTkwas predicted as a probable sugar
isomerase related to the C-terminal sugar isomerase domain of GlcN6P synthase, the recombinant GlmDTk
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
GlmDTk. In T. kodakaraensis cells, glmDTkwas polycistronically transcribed together with upstream genes
encoding an ABC transporter and a downstream exo-?-glucosaminidase gene (glmATk) within the gene cluster,
and their expression was induced by the chitin degradation intermediate, diacetylchitobiose. The results
presented here indicate that GlmDTkis 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.
Amino sugars, such as N-acetylglucosamine (GlcNAc), N-
acetylgalactosamine (GalNAc), and N-acetylmuramic acid, are
important building blocks for structural polysaccharides or
sugar chains in several organisms. In the metabolism of these
sugars, the conversion between fructose-6-phosphate (Fru6P)
and glucosamine-6-phosphate (GlcN6P) is a key step in both
anabolic and catabolic directions. The anabolic reaction is cat-
alyzed by GlcN6P synthase (L-glutamine:D-fructose-6-phos-
phate amidotransferase), while catabolism is mediated by
GlcN6P deaminase (Fig. 1A). GlcN6P synthase catalyzes the
irreversible formation of GlcN6P and glutamate from Fru6P
and glutamine and is classified in a glutamine-dependent
amidotransferase family (18) comprised of an N-terminal glu-
tamine amide transfer (GAT) domain joined to a C-terminal
sugar isomerase domain (Fig. 1B). The former domain pro-
duces ammonia from glutamine, and the generated ammonia is
utilized for amination of Fru6P accompanied by isomerization
to GlcN6P in the latter domain. Unlike other glutamine-de-
pendent amidotransferases displaying ammonia-dependent ac-
tivity, GlcN6P synthase cannot utilize free ammonia as the
nitrogen donor in place of glutamine (19).
On the other hand, GlcN6P deaminase catalyzes the deami-
nation-isomerization reaction from GlcN6P to Fru6P and am-
monia and can also catalyze its reverse reaction under the
presence of high concentrations of ammonia (7, 20). Although
GlcN6P synthase and GlcN6P deaminase catalyze similar re-
actions, there is no relation between the primary structures of
these two enzymes. There have been many studies on GlcN6P
synthase (3, 5, 13, 24) and GlcN6P deaminase (1, 8, 15–17, 20)
from Eucarya and Bacteria due to the importance of these
enzymes in the regulation of amino sugar metabolism. In con-
trast, corresponding enzymes from Archaea have not been re-
ported so far. Most intriguingly, archaeal genomes do not
harbor any genes homologous to known GlcN6P deaminases.
We have previously found that the hyperthermophilic ar-
chaeon Thermococcus kodakaraensis KOD1 (2) has an ability
to degrade chitin, a ?-1,4-linked linear homopolymer of
GlcNAc, and successfully identified a novel chitin catabolic
pathway. Namely, chitin is first degraded into the disaccharide
GlcNAc2by a unique extracellular chitinase from T. koda-
karaensis (ChiATk) possessing endo- and exo-type catalytic do-
mains (25, 28). The GlcNAc2is probably translocated across
the cell membrane by an ABC transport system and then
deacetylated by a deacetylase (DacTk) with nonreducing end
specificity. The partially acetylated disaccharide GlcN-GlcNAc
is hydrolyzed into GlcN and GlcNAc by an exo-?-glucosamini-
dase (GlmATk), and the generated GlcNAc is further deacety-
lated to GlcN by DacTk(26, 27), resulting in the complete
* Corresponding author. Department of Synthetic Chemistry and
Biological Chemistry, Graduate School of Engineering, Kyoto Univer-
sity, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan. Phone: 81-75-383-
2777. Fax: 81-75-383-2778. E-mail: email@example.com.
† Present address: Nanotechnology Research Institute, National In-
stitute 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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7044TANAKA ET AL.J. BACTERIOL.