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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

	ABSTRACT

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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.

	INTRODUCTION

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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 catalyzed by GlcN6P synthase (L-glutamine:D-fructose-6-phosphate 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 glutamine amide transfer (GAT) domain joined to a C-terminal sugar isomerase domain (Fig. 1B). The former domain produces 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-dependent amidotransferases displaying ammonia-dependent activity, GlcN6P synthase cannot utilize free ammonia as the nitrogen donor in place of glutamine (19).

View larger version (27K): [in this window] [in a new window]   FIG. 1. (A) Reactions catalyzed by GlcN6P deaminase and GlcN6P synthase. (B) Schematic diagrams of the domain structures of known GlcN6P deaminases and GlcN6P synthases. (C) Schematic diagrams of the domain structures of GlmDTk and GlmSTk identified on the T. kodakaraensis genome.

We have previously found that the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (2) has an ability to degrade chitin, a ß-1,4-linked linear homopolymer of GlcNAc, and successfully identified a novel chitin catabolic pathway. Namely, chitin is first degraded into the disaccharide GlcNAc₂ by a unique extracellular chitinase from T. kodakaraensis (ChiA_Tk) possessing endo- and exo-type catalytic domains (25, 28). The GlcNAc₂ 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-ß-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. AP006878) (12). Here, we focused on a gene within this cluster, encoding a probable sugar isomerase related to the isomerase domain of GlcN6P synthase, and clearly demonstrated that this probable sugar isomerase exhibited GlcN6P deaminase activity. This report identifies not only the first archaeal GlcN6P deaminase involved in chitin degradation but also a novel type of GlcN6P deaminase distinct from previously known enzymes.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and media. T. kodakaraensis KOD1 was grown anaerobically at 85°C in a screw-cap bottle with MA medium (4.8 g and 26.4 g of Marine Art SF agents A and B, respectively [Senju Seiyaku, Osaka, Japan], 5 g of yeast extract, and 5 g of tryptone in 1 liter of deionized water) supplemented with elemental sulfur (5 g/liter). Escherichia coli DH5 and BL21-CodonPlus(DE3)-RIL (Stratagene, La Jolla, CA) were used as hosts for the expression plasmid derived from pET-21a (Novagen, Madison, WI) and were cultivated in LB medium at 37°C.

DNA manipulations and sequencing. DNA manipulations were carried out by standard methods, as described previously by Sambrook and Russell (23). Restriction enzymes and other modifying enzymes were purchased from Takara Bio (Otsu, Shiga, Japan) or Toyobo (Osaka, Japan). Small-scale preparation of plasmid DNA from E. coli cells was performed with the QIAGEN plasmid mini kit (QIAGEN, Hilden, Germany). DNA sequencing was performed with the BigDye Terminator cycle sequencing ready reaction kit, version 3.1, and the model 3100 capillary DNA sequencer (Applied Biosystems, Foster City, CA).

Construction of the expression plasmid. The expression plasmid for glmD_Tk was constructed by PCR as described below. Two oligonucleotides (sense, 5'-GGTGAGCATATGCACGCAACGCTTAGAG-3', and antisense, 5'-CCGGATCCCATCACCACTTTACGAC-3'[Underlined sequences indicate an NdeI site in the sense primer and a BamHI site in the antisense primer.]) and T. kodakaraensis genomic DNA were used as the primers and the template for DNA amplification, respectively. The amplified DNA was digested with NdeI and BamHI and then ligated with the corresponding sites in the plasmid pET-21a. The absence of unintended mutations in the insert was confirmed by DNA sequencing. The resulting plasmid was designated pET-glmD.

Purification of recombinant GlmD_Tk. E. coli BL21-CodonPlus(DE3)-RIL cells harboring pET-glmD were induced for overexpression with 0.1 mM isopropyl-ß-D-thiogalactopyranoside at the mid-exponential growth phase and incubated for a further 4 h at 37°C. The cells were harvested by centrifugation (5,000 x g for 10 min at 4°C), resuspended in buffer A (50 mM Tris-HCl [pH 8.0]), and then disrupted by sonication. The supernatant after centrifugation (15,000 x g for 10 min) was incubated at 85°C for 10 min and centrifuged (15,000 x g for 10 min) to obtain a heat-stable protein solution. The solution was applied to an anion-exchange Resource Q column (6 ml) (Amersham Biosciences, Piscataway, NJ) equilibrated with buffer A. The proteins were eluted with a linear gradient of 0 to 0.5 M NaCl, and the peak fractions eluted at 0.3 M NaCl were concentrated using an Ultrafree-4 centrifugal filter unit Biomax-10 (Millipore, Bedford, MA). This was applied to a gel filtration Superdex-200 HR 10/30 column (Amersham Biosciences) equilibrated with buffer A containing 0.15 M NaCl. Protein concentration was determined with the Bio-Rad protein assay (Bio-Rad, Hercules, CA), with bovine serum albumin as a standard.

Enzyme assays. A qualitative assay for GlmD_Tk toward various monosaccharides was performed with silica gel thin-layer chromatography (TLC) as described previously (25), with a modification in the developer to methanol-chloroform-acetic acid-water (30:20:10:1 [vol/vol/vol/vol]). For detection of the products, aniline-diphenylamine reagent and ninhydrin reagent were applied.

GlcN6P deaminase activity was determined by a coupled enzymatic assay with phosphoglucose isomerase from baker's yeast (Nacalai Tesque, Kyoto, Japan) and glucose-6-phosphate (Glc6P) dehydrogenase from Leuconostoc mesenteroides (Sigma, St. Louis, Mo.). The reaction mixture (30 µl), containing 3.33 mM GlcN6P and 50 ng GlmD_Tk in 50 mM CHES [2-(N-cyclohexylamino)ethanesulfonic acid]-NaOH(adjusted to pH 8.3 at 60°C), was incubated for 1 min at 60°C. After terminating the reaction by rapid cooling, 270 µl of coupling reaction mixture (0.56 mM NAD⁺, 0.6 U phosphoglucose isomerase, 0.25 U Glc6P dehydrogenase in 47.8 mM Tris-HCl [pH 8.0]) was added and was then incubated for 30 min at 25°C. Absorbance at 340 nm derived from NADH formation was measured spectrophotometrically. To determine the optimal temperature, the first reaction was performed at various temperatures (25 to 100°C). The optimal pH was determined by measuring the activity using the following buffers: MES [Z-(N-morpholino)ethanesulfonic acid]-NaOH (pH 5.5 to 7.0), Tris-HCl (pH 7.0 to 8.5), bicine-NaOH (pH 7.5 to 9.0), and CHES-NaOH (pH 8.5 to 10.5). The pH values were adjusted at room temperature, and the values at higher temperatures were calculated according to the temperature coefficients for the respective buffers (9). To investigate the thermostability of GlmD_Tk, the enzyme solution (50 ng GlmD_Tk in 20 µl of 75 mM CHES-NaOH [pH 8.3 at 60°C]) was incubated at 80°C or 90°C from 5 to 100 min before the first reaction, and the resulting activity was determined by the method described above. The activity observed prior to incubation was 100%.

The reverse reaction of GlcN6P deaminase was determined by measuring the generated GlcN6P. The reaction mixture (100 µl), containing 5 mM Fru6P, 10 mM NH₄Cl, and 150 ng GlmD_Tk in 50 mM CHES-NaOH (pH 8.3 at 60°C), was incubated for 1 min at 60°C. The reaction was terminated by cooling and followed by filtration with a Microcon YM-10 (Millipore) to remove the enzyme. The amount of GlcN6P in the filtrate was determined by a modified method of Morgan and Elson (13).

RNA experiments. For the isolation of RNA from T. kodakaraensis KOD1, cells grown in MA medium with or without 0.1% GlcNAc₂ or maltose were harvested at the early exponential growth phase when A₆₆₀ was around 0.18. Total RNA was isolated using the RNeasy Midi kit and RNase-Free DNase set (QIAGEN). For reverse transcription (RT)-PCR, 40 ng of total RNA from the cells grown with GlcNAc₂ was used with the ThermoScript RT-PCR system (Invitrogen, Carlsbad, CA). Oligonucleotides RT1, RT2, and RT3 were used for the RT reaction, and pairs of oligonucleotides, F1-R1, F2-R2, and F3-R3, were used for successive PCR amplification. The sequences are as follows: RT1, 5'-CAACCTCCTGCTGAAAG-3'; RT2, 5'-GCTAAACTCAACCTTTCC-3'; RT3, 5'-CCTAACGTCAAGAATTG-3'; F1, 5'-GGTGAGTGAATGCACGC-3'; R1, 5'-CCTCATCGTGGTTGCAG-3'; F2, 5'-GGAGGGTTGAAAATGGC-3'; R2, 5'-CATCACCACTTTACGAC-3'; F3, 5'-GGTGTCCATGAAGAAAGC-3'; and R3, 5'-GTCATTCGCCACCCCTC-3'. For Northern blot analysis, 30 µg of total RNA from the cells grown with GlcNAc₂ was separated by denaturing agarose gel electrophoresis and transferred to positively charged nylon membranes (Roche Diagnostics, Basel, Switzerland) by capillary blotting. For RNA dot blot hybridization analysis, 30 µg of total RNA (3 µl) was dropped onto the membrane and was immobilized by UV cross-linking. DNA fragments of approximately 600 bp within the coding regions of glmD_Tk, glmS_Tk, and a DNA ligase gene (lig_Tk) were amplified by PCR and used as a template for probe preparation. The sequences of the primer pairs were as follows: F4, 5'-GTTCCGAAATTTGCCCTG-3', and R4, 5'-GATGGCTTTATAGTATGAG-3', for glmD_Tk; F5, 5'-GTCGAAGAGGCGAGCGAG-3', and R5, 5'-CGCTCGGGTTTATTGCAAC-3', for glmS_Tk; and F6, 5'-GAAGAGCTTCTTCTCACAGCC-3', and R6, 5'-CAAGCTTATTCCTCCTGTCG-3', for lig_Tk. Digoxigenin labeling of DNA fragments, hybridization, and detection of signals were performed according to the instructions of the manufacturer (Roche Diagnostics).

Western blot analysis. T. kodakaraensis KOD1 was cultivated in 10 ml of MA medium supplemented with various kinds of saccharides (final concentration, 0.5%) and 20 µl of polysulfide solution (20% elemental sulfur in 3 M Na₂S) in place of elemental sulfur. The cells were harvested, disrupted by sonication in buffer A containing protein inhibitor mix (Complete Mini; Roche Diagnostics) and then centrifuged (15,000 x g for 30 min) to obtain soluble fractions. Each fraction was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and successive Western blot analysis using specific antiserum (rabbit) against the recombinant GlmD_Tk. A protein A-peroxidase conjugate was used to visualize the specific protein together with 4-chloro-1-naphthol and hydrogen peroxide.

Nucleotide sequence accession number. The nucleotide sequence data of the glmD_Tk gene reported here (TK1755) have been included within the T. kodakaraensis KOD1 genome (accession number AP006878) in the EMBL, GenBank, and DDBJ nucleotide sequence databases.

	RESULTS

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A probable sugar isomerase gene within the chitin degradation gene cluster in T. kodakaraensis. We have previously reported that the genes for chitin catabolic enzymes chitinase (ChiA_Tk encoded by TK1765) (25, 28), GlcNAc₂ deacetylase (Dac_Tk encoded by TK1764) (27), and ß-glucosaminidase (GlmA_Tk encoded by TK1754) (26) are clustered at the 1,553- to 1,569-kbp region of the T. kodakaraensis KOD1 genome (12), as shown in Fig. 2. In this cluster, one gene (TK1755) was identified between the glmA_Tk and putative ABC transporter genes (TK1756 to TK1760) in the same orientation. As these seven genes were overlapping or separated by short interval regions (from –8 bp to 47 bp), they were supposed to be transcribed into a single mRNA. The TK1755 gene, designated glmD_Tk, consisted of 978 bp encoding a protein of 326 amino acids with a predicted molecular mass of 36,749 Da. The deduced amino acid sequence displayed high overall homologies to the proteins from the closely related hyperthermophilic archaea Pyrococcus furiosus (PF0362), Pyrococcus abyssi (PAB1348), and Pyrococcus horikoshii (PH0510) (61 to 63% identical at the amino acid level). GlmD_Tk also showed weak but notable similarities to the C-terminal isomerase domain of GlcN6P synthases from various sources (25 to 32% identities), although it lacks an N-terminal GAT domain (Fig. 1C). The GlmD_Tk-related proteins and domains commonly contained a tandem repeat of two sugar isomerase subdomains (SIS domain, Pfam01380). Previous X-ray crystallographic analyses for GlcN6P synthase from Escherichia coli (GlmS_Ec) have identified catalytically important amino acid residues, Glu-488, His-504, and Lys-603, for the amination and isomerization of Fru6P (30, 31). We found that the corresponding residues were also conserved in GlmD_Tk as Glu-214, His-230, and Lys-322, suggesting a sugar isomerization activity in GlmD_Tk with a catalytic mechanism similar to that of GlcN6P synthase. It should be noted that the T. kodakaraensis genome harbors a separate gene (designated glmS_Tk) encoding a protein entirely homologous to GlcN6P synthase (Fig. 1C), composed of GAT and isomerase domains, at a different locus (TK0809). The C-terminal isomerase domain showed 27% identity to GlmD_Tk.

View larger version (18K): [in this window] [in a new window]   FIG. 2. Gene organization in the 23.7-kbp region, including glmDTk, on the T. kodakaraensis genome. Arrows indicate open reading frames, and their translation products are indicated above the arrows. Black arrow, glmDTk (TK1755); gray arrows, glmATk (TK1754) (26), glyTk (TK1761) (11), dacTk (TK1764) (27), and chiATk (TK1765) (25, 28), which have been characterized previously; white arrows, uncharacterized open reading frames. Oligonucleotides used for the RT-PCR experiment are also indicated as small arrows at the bottom.

Production and purification of recombinant GlmD_Tk. To investigate the function of the protein product of glmD_Tk, the gene was expressed in E. coli with the pET expression system. The recombinant protein was obtained in a soluble form and was purified to apparent homogeneity in SDS-PAGE by heat treatment and column chromatography (Fig. 3A). The molecular mass of the recombinant GlmD_Tk was estimated to be 37.3 kDa by SDS-PAGE and 71.8 kDa by gel filtration chromatography, indicating formation of a homodimer. This dimeric structure was the same as that of the typical GlcN6P synthase but differed from the general homohexameric structure of GlcN6P deaminases. Some glutamine-dependent amidotransferases have been known to possess a GAT domain as a distinct subunit (18). We therefore examined the possibility of heteromeric association of GlmD_Tk with other proteins in T. kodakaraensis cells by native PAGE Western blot analysis, as shown in Fig. 3B. GlmD_Tk was actually expressed in the cells grown in the presence of GlcNAc₂, and the mobility of native GlmD_Tk coincided with that of the purified, recombinant protein. This result indicated that GlmD_Tk was present in this archaeon as a homodimeric enzyme without any other subunits.

View larger version (70K): [in this window] [in a new window]   FIG. 3. (A) SDS-PAGE results for samples through the purification steps of recombinant GlmDTk from E. coli. Lane 1, cell extract of E. coli before induction; lane 2, cell extract after induction for 4 h; lane 3, soluble fraction after sonication; lane 4, thermostable protein fraction after heat treatment at 85°C for 10 min; lane 5, peak fraction after anion-exchange chromatography; lane 6, peak fraction after gel filtration chromatography; lane M, molecular mass marker. In lanes 3 to 6, 5 µg of proteins was applied. (B) Western blot analysis results after native PAGE of recombinant GlmDTk and cell extracts of T. kodakaraensis. For native PAGE, a 5 to 20% gradient gel was used. Lane 1, recombinant GlmDTk purified from E. coli (50 ng); lane 2, cell extracts of T. kodakaraensis grown in 0.1% GlcNAc2-containing medium for 24 h (30 µg); lane M, molecular mass marker.

View larger version (66K): [in this window] [in a new window]   FIG. 4. Catalytic activity of GlmDTk against GlcN6P or Fru6P determined by TLC analysis. The reaction mixture (10 µl) containing 40 mM of the respective component(s) described in the figure was incubated with 0.4 µg of GlmDTk at 60°C for 60 min. The TLC plate was visualized with an aniline-diphenylamine reagent. std., standard containng 10 µg of authentic GlcN6P and Fru6P.

View larger version (25K): [in this window] [in a new window]   FIG. 5. (A) Three overlapped segmental RT-PCR analyses of the region for the genes of the ABC transporter, glmDTk and glmATk. Annealing sites for oligonucleotides used for the RT reaction and PCR are indicated in Fig. 2. Lanes 1 and 4, amplification with RT3, F3, and R3 (3,899 bp); lanes 2 and 5, amplification with RT2, F2, and R2 (3,874 bp); lanes 3 and 6, amplification with RT1, F1, and R1 (3,513 bp); lane M, molecular mass marker. Lanes 4 to 6 are the results of negative controls without the RT reactions. (B) Dot blot analysis of RNA from T. kodakaraensis grown with or without 0.1% GlcNAc2 or maltose using specific probes against ligTk, glmDTk, and glmSTk genes. Each spot contains 30 µg of total RNA. (C) Western blot analysis of the cell extracts of T. kodakaraensis KOD1 grown in media containing various sugars (0.5%) with elemental sulfur at 85°C for 24 h. The amount of protein applied was 20 µg (T. kodakaraensis cell extract) or 50 ng (recombinant GlmDTk, lane C).

	DISCUSSION

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In this study, we revealed that one gene, TK1755, in the gene cluster for chitin degradation on the T. kodakaraensis genome, encoded a GlcN6P deaminase structurally distinct from previously known deaminases. The protein product (GlmD_Tk) displayed similarity to the sugar isomerase domain of GlcN6P synthases. GlmD_Tk kinetically preferred the deamination of GlcN6P rather than the reverse ammonia-dependent amination of Fru6P (Table 1), supporting the function of this enzyme in amino sugar catabolism in vivo. Based on these facts together with our previous results (25-28), we can now clearly envision the chitin metabolism in this hyperthermophile in its near entirety, as summarized in Fig. 6. GlmD_Tk was estimated to have a role in the final step to allow entrance of the chitin-derived catabolites into glycolysis. The highly clustered genes for this pathway were transcriptionally induced by GlcNAc₂ generated from chitin. Although this gene cluster lacks genes for the phosphorylation of GlcN, it is likely that this step can be mediated by an ADP-dependent glucokinase encoded at a different locus (TK1110), as the orthologs from the closely related archaea P. furiosus and Thermococcus litoralis have been reported to be capable of phosphorylating GlcN as well as glucose (14).

View larger version (23K): [in this window] [in a new window]   FIG. 6. Proposed chitin catabolic pathway in T. kodakaraensis KOD1 leading to a glycolytic intermediate.

Besides the isomerase domain of GlcN6P synthase, GlmD_Tk shares overall homology against proteins existing in a subset of Archaea and Bacteria. These homologs are also comprised of tandem SIS domains and classified as COG2222 in the Cluster of Orthologous Group of proteins database. Among them, the Pyrococcus homologs not only showed high homology to glmD_Tk but also were commonly located within the completely conserved gene cluster from dac to glmA for GlcNAc₂ catabolism. Although separated into two open reading frames at a different locus, P. furiosus harbors chitinase genes similar to ChiA_Tk. Therefore, the cluster in P. furiosus is likely to be involved in chitin degradation, as in the case of T. kodakaraensis. Interestingly, P. abyssi and P. horikoshii also harbor this gene cluster despite the absence of a chitinase ortholog. We have previously reported that Gly_Tk, which displays broad specificity to various ß-1,4-glycosides (11), is encoded in this cluster (Fig. 2). The enzyme does not seem to participate in GlcNAc₂ metabolism (26), but the induction behavior was the same as those of the other clustered genes for chitinolysis (27). Although the physiological role of this ß-glycosidase will have to be clarified, the common presence of the gene cluster, containing gly in P. abyssi and P. horikoshii, suggests an additional role of this cluster among members of the Thermococcales order. The cluster may be involved in the degradation of other cellular ß-1,4-linked heteropolysaccharides. The proteins classified into COG2222 include AgaS encoded within the GalNAc catabolic cluster in E. coli. It has been reported that the bacterial metabolism of GalNAc was similar to that of GlcNAc (4, 22). In this pathway, galactosamine-6-phosphate (GalN6P) was predicted to be converted to tagatose-6-phosphate by the function of AgaI corresponding to the classical GlcN6P deaminase NagB. Although the catalytic property of AgaS has not been investigated yet, our results raise the possibility that AgaS might be functional as an additional GalN6P deaminase to achieve efficient processing of GalNAc.

In this study, we identified and characterized a new type of GlcN6P deaminase from the hyperthermophilic archaeon T. kodakaraensis and hereby provided an overview of the chitin catabolic pathway to glycolysis in this organism. As the enzymes in this pathway, ChiA_Tk, GlmD_Tk, Dac_Tk, and GlmA_Tk, were all novel enzymes with high thermostability, it is expected that these enzymes can be useful catalysts for future conversion of the unused biomass chitin.

	ACKNOWLEDGMENTS

 
This work was supported by a Grant-in-Aid for Scientific Research (no. 14103011) to T.I. from the Japan Society for the Promotion of Science (JSPS) and supported in part by a Grant-in-Aid for JSPS fellows (no. 15 · 02342) to T.T. from the Ministry of Education, Science, Sports, Culture, and Technology.

	FOOTNOTES

 
* Corresponding author. Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan. Phone: 81-75-383-2777. Fax: 81-75-383-2778. E-mail: imanaka{at}sbchem.kyoto-u.ac.jp.

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.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Arreola, R., B. Valderrama, M. L. Morante, and E. Horjales.2003 . Two mammalian glucosamine-6-phosphate deaminases: a structural and genetic study. FEBS Lett. 551:63-70.[CrossRef][Medline]
2. Atomi, H., T. Fukui, T. Kanai, M. Morikawa, and T. Imanaka.2004 . Description of Thermococcus kodakaraensis sp. nov., a well studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1. Archaea 1:263-267.[Medline]
3. Badet-Denisot, M. A., L. A. Fernandez-Herrero, J. Berenguer, T. Ooi, and B. Badet. 1997. Characterization of L-glutamine:D-fructose-6-phosphate amidotransferase from an extreme thermophile Thermus thermophilus HB8. Arch. Biochem. Biophys. 337:129-136.[CrossRef][Medline]
4. Brinkkötter, A., H. Klöß, C. Alpert, and J. W. Lengeler.2000 . Pathways for the utilization of N-acetyl-galactosamine and galactosamine in Escherichia coli.Mol. Microbiol. 37:125-135.[CrossRef][Medline]
5. Broschat, K. O., C. Gorka, J. D. Page, C. L. Martin-Berger, M. S. Davies, H. -C. Huang, E. A. Gulve, W. J. Salsgiver, and T. P. Kasten. 2002. Kinetic characterization of human glutamine-fructose-6-phosphate amidotransferase I: potent feedback inhibition by glucosamine 6-phosphate. J. Biol. Chem. 277:14764-14770.[Abstract/Free Full Text]
6. Bustos-Jaimes, I., A. Sosa-Peinado, E. Rudiño-Piñera, E. Horjales, and M. L. Calcagno. 2002. On the role of the conformational flexibility of the active-site lid on the allosteric kinetics of glucosamine-6-phosphate deaminase. J. Mol. Biol. 319:183-189.[CrossRef][Medline]
7. Calcagno, M., P. J. Campos, G. Mulliert, and J. Suástegui.1984 . Purification, molecular and kinetic properties of glucosamine-6-phosphate isomerase (deaminase) from Escherichia coli. Biochim. Biophys. Acta 787:165-173.[CrossRef][Medline]
8. Comb, D. G., and S. Roseman. 1958. Glucosamine metabolism. IV. Glucosamine-6-phosphate deaminase. J. Biol. Chem. 232:807-827.[Free Full Text]
9. Dawson, R. M. C., D. C. Elliott, W. H. Elliott, and K. M. Jones. 1986. Data for biochemical research, 3rd ed., p. 417-448. Clarendon Press, Oxford, England.
10. Denisot, M. A., F. Le Goffic, and B. Badet. 1991. Glucosamine-6-phosphate synthase from Escherichia coli yields two proteins upon limited proteolysis: identification of the glutamine amidohydrolase and 2R ketose/aldose isomerase-bearing domains based on their biochemical properties. Arch. Biochem. Biophys. 288:225-230.[CrossRef][Medline]
11. Ezaki, S., K. Miyaoku, K. Nishi, T. Tanaka, S. Fujiwara, M. Takagi, H. Atomi, and T. Imanaka. 1999. Gene analysis and enzymatic properties of thermostable ß-glycosidase from Pyrococcus kodakaraensis KOD1. J. Biosci. Bioeng. 88:130-135.
12. Fukui, T., H. Atomi, T. Kanai, R. Matsumi, S. Fujiwara, and T. Imanaka.2005 . Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes. Genome Res. 15:352-363.[Abstract/Free Full Text]
13. Ghosh, S., H. J. Blumenthal, E. Davidson, and S. Roseman.1960 . Glucosamine metabolism. V. Enzymatic synthesis of glucosamine 6-phosphate. J. Biol. Chem. 235:1265-1273.[Free Full Text]
14. Koga, S., I. Yoshioka, H. Sakuraba, M. Takahashi, S. Sakasegawa, S. Shimizu, and T. Ohshima. 2000. Biochemical characterization, cloning, and sequencing of ADP-dependent (AMP-forming) glucokinase from two hyperthermophilic archaea, Pyrococcus furiosus and Thermococcus litoralis. J. Biochem. (Tokyo) 128:1079-1085.[Abstract/Free Full Text]
15. Lara-Gonzalez, S., H. B. Dixon, G. Mendoza-Hernández, M. M. Altamirano, and M. L. Calcagno. 2000. On the role of the N-terminal group in the allosteric function of glucosamine-6-phosphate deaminase from Escherichia coli.J. Mol. Biol. 301:219-227.[CrossRef][Medline]
16. Lara-Lemus, R., and M. L. Calcagno. 1998. Glucosamine-6-phosphate deaminase from beef kidney is an allosteric system of the V-type. Biochim. Biophys. Acta 1388:1-9.[CrossRef][Medline]
17. Lara-Lemus, R., C. A. Libreros-Minotta, M. M. Altamirano, and M. L. Calcagno. 1992. Purification and characterization of glucosamine-6-phosphate deaminase from dog kidney cortex. Arch. Biochem. Biophys. 297:213-220.[CrossRef][Medline]
18. Massiére, F., and M. A. Badet-Denisot. 1998. The mechanism of glutamine-dependent amidotransferases. Cell. Mol. Life Sci. 54:205-222.[CrossRef][Medline]
19. Milewski, S. 2002. Glucosamine-6-phosphate synthase—the multi-facets enzyme. Biochim. Biophys. Acta 1597:173-192.[CrossRef][Medline]
20. Natarajan, K., and A. Datta. 1993. Molecular cloning and analysis of the NAG1 cDNA coding for glucosamine-6-phosphate deaminase from Candida albicans. J. Biol. Chem. 268:9206-9214.[Abstract/Free Full Text]
21. Oliva, G., M. R. Fontes, R. C. Garratt, M. M. Altamirano, M. L. Calcagno, and E. Horjales.1995 . Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 Å resolution. Structure 3:1323-1332.[Medline]
22. Reizer, J., T. M. Ramseier, A. Reizer, A. Charbit, and M. H. Saier, Jr. 1996. Novel phosphotransferase genes revealed by bacterial genome sequencing: a gene cluster encoding a putative N-acetylgalactosamine metabolic pathway in Escherichia coli. Microbiology 142:231-250.[Abstract]
23. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
24. Smith, R. J., S. Milewski, A. J. Brown, and G. W. Gooday. 1996. Isolation and characterization of the GFA1 gene encoding the glutamine:fructose-6-phosphate amidotransferase of Candida albicans. J. Bacteriol. 178:2320-2327.[Abstract/Free Full Text]
25. Tanaka, T., S. Fujiwara, S. Nishikori, T. Fukui, M. Takagi, and T. Imanaka.1999 . A unique chitinase with dual active sites and triple substrate binding sites from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1. Appl. Environ. Microbiol. 65:5338-5344.[Abstract/Free Full Text]
26. Tanaka, T., T. Fukui, H. Atomi, and T. Imanaka. 2003. Characterization of an exo-ß-D-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J. Bacteriol. 185:5175-5181.[Abstract/Free Full Text]
27. Tanaka, T., T. Fukui, S. Fujiwara, H. Atomi, and T. Imanaka.2004 . Concerted action of diacetylchitobiose deacetylase and exo-ß-D-glucosaminidase in a novel chitinolytic pathway in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J. Biol. Chem. 279:30021-30027.[Abstract/Free Full Text]
28. Tanaka, T., T. Fukui, and T. Imanaka. 2001. Different cleavage specificities of the dual catalytic domains in chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.J. Biol. Chem. 276:35629-35635.[Abstract/Free Full Text]
29. Teplyakov, A., G. Obmolova, B. Badet, and M. A. Badet-Denisot.2001 . Channeling of ammonia in glucosamine-6-phosphate synthase. J. Mol. Biol. 313:1093-1102.[CrossRef][Medline]
30. Teplyakov, A., G. Obmolova, M. A. Badet-Denisot, and B. Badet.1999 . The mechanism of sugar phosphate isomerization by glucosamine 6-phosphate synthase. Protein Sci. 8:596-602.[Abstract]
31. Teplyakov, A., G. Obmolova, M. A. Badet-Denisot, B. Badet, and I. Polikarpov. 1998. Involvement of the C terminus in intramolecular nitrogen channeling in glucosamine 6-phosphate synthase: evidence from a 1.6 Å crystal structure of the isomerase domain. Structure 6:1047-1055.[Medline]

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