Concerted Action of Diacetylchitobiose Deacetylase and Exo-β-D-glucosaminidase in a Novel Chitinolytic Pathway in the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1
2004; Elsevier BV; Volume: 279; Issue: 29 Linguagem: Inglês
10.1074/jbc.m314187200
ISSN1083-351X
AutoresTakeshi Tanaka, Toshiaki Fukui, Shinsuke Fujiwara, Haruyuki Atomi, Tadayuki Imanaka,
Tópico(s)Peptidase Inhibition and Analysis
ResumoThe hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 possesses chitinase (Tk-ChiA) and exo-β-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-α-d-glucopyranoside deacetylase. Recombinant Tk-Dac showed deacetylase activity toward N-acetylchitooligosaccharides (GlcNAc2–5), 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. The hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 possesses chitinase (Tk-ChiA) and exo-β-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-α-d-glucopyranoside deacetylase. Recombinant Tk-Dac showed deacetylase activity toward N-acetylchitooligosaccharides (GlcNAc2–5), 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. Chitin is the linear homopolymer of β-1,4-linked N-acetylglucosamine (GlcNAc), and its biological production is the most abundant after cellulose. Degradation of chitin in eucaryotes and bacteria has been studied very well, and chitin catabolic pathways clarified from these studies are summarized in Fig. 1A (thin arrows). Chitin is degraded into diacetylchitobiose (GlcNAc2) by the combination of endo- and exo-type chitinases (reactions 1 and 2) followed by dimer processing with β-N-acetylglucosaminidase (GlcNAcase 1The abbreviations used are: GlcNAcase, β-N-acetylglucosaminidase; CHES, 2-(cyclohexylamino)ethanesulfonic acid; MES, 4-morpholineethanesulfonic acid; GlcNAc2, diacetylchitobiose; GlcNAc6P, GlcNAc 6-phosphate; GlcNase, exo-β-d-glucosaminidase; Tk, T. kodakaraensis; GlcN2, chitobiose; GlcNAc-4MU, 4-methylumbelliferyl N-acetyl-β-d-glucosaminide; RT, reverse transcription; TEMED, N,N,N′,N′-tetramethylethylene-diamine; GlcNAc, N-acetylglucosamine; GlcN, glucosamine.; reaction 3), GlcNAc2 phosphorylase (reaction 4), or GlcNAc2 phosphotransferase system (reaction 5) and 6-phospho-β-glucosaminidase (reaction 6) (1Gooday G.W. Ratledge C. Biochemistry of Microbial Degradation. Kluwer Academic Publishers, Dordrecht, Netherlands1994: 279-312Crossref Google Scholar, 2Park J.K. Keyhani N.O. Roseman S. J. Biol. Chem. 2000; 275: 33077-33083Abstract Full Text Full Text PDF PubMed Google Scholar, 3Keyhani N.O. Roseman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14367-14371Crossref PubMed Scopus (86) Google Scholar, 4Keyhani N.O. Wang L.X. Lee Y.C. Roseman S. J. Biol. Chem. 2000; 275: 33084-33090Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In these pathways removal of the N-acetyl group derived from the starting chitin occurs after the degradation to monomers. This step is catalyzed by GlcNAc-6-phosphate (GlcNAc6P) deacetylase, which deacetylates the GlcNAc6P produced by cleavage of GlcNAc6P-GlcNAc (reaction 6) or by phosphorylation of GlcNAc. An alternative pathway for chitin degradation is proposed to be initiated by deacetylation of chitin by chitin deacetylase (reaction 7). The resulting deacetylated chitin, chitosan, is then degraded to glucosamine (GlcN) by chitosanase (endo-type enzyme; reaction 8) in cooperation with exo-β-d-glucosaminidase (GlcNase; reaction 9) (1Gooday G.W. Ratledge C. Biochemistry of Microbial Degradation. Kluwer Academic Publishers, Dordrecht, Netherlands1994: 279-312Crossref Google Scholar). In contrast to eucaryotes and bacteria, there is very little information concerning chitinolysis in archaea. We previously reported the first characterization of a thermostable chitinase from a hyperthermophilic archaeon, Thermococcus kodak-araensis KOD1 (5Tanaka T. Fujiwara S. Nishikori S. Fukui T. Takagi M. Imanaka T. Appl. Environ. Microbiol. 1999; 65: 5338-5344Crossref PubMed Google Scholar, 6Tanaka T. Fukui T. Imanaka T. J. Biol. Chem. 2001; 276: 35629-35635Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The chitinase (Tk-ChiA) has a unique domain structure that is composed of dual catalytic domains and triple chitin binding domains. The dual catalytic domains can individually cleave chitin chains with different profiles, namely, the N- and C-terminal catalytic domains exhibit exo- and endo-type cleavage specificities, respectively. Other archaeal chitinases from the hyperthermophiles Thermococcus chitonophagus (7Andronopoulou E. Vorgias C.E. Extremophiles. 2003; 7: 43-53Crossref PubMed Scopus (40) Google Scholar) and Pyrococcus furiosus (8Gao J. Bauer M.W. Shockley K.R. Pysz M.A. Kelly R.M. Appl. Environ. Microbiol. 2003; 69: 3119-3128Crossref PubMed Scopus (76) Google Scholar) have also been reported. These chitinases from hyperthermophilic archaea, including Tk-ChiA, produce GlcNAc2 as an end product from chitin. Recently, we have identified another chitinolytic enzyme, GlcNase, from T. kodakaraensis in the course of searching for enzymes involved in GlcNAc2 catabolism (9Tanaka T. Fukui T. Atomi H. Imanaka T. J. Bacteriol. 2003; 185: 5175-5181Crossref PubMed Scopus (89) Google Scholar). This GlcNase (Tk-GlmA) hydrolyzed chitobiose (GlcN2) to GlcN and was induced by GlcNAc2, the end product from chitin by Tk-ChiA. These facts have suggested the presence of a GlcNAc2-specific deacetylase activity in T. kodakaraensis in order to supply the substrate for Tk-GlmA from GlcNAc2 (Fig. 1A, thick arrows). In this study we performed purification and characterization of a GlcNAc2 deacetylase (Tk-Dac) from T. kodakaraensis and have clarified that one gene with previously unknown function encoded this protein. Tk-Dac deacetylated the nonreducing terminal unit of N-acetylchitooligosaccharides as well as GlcNAc monomers, constituting a novel archaeal chitin catabolic pathway in this organism together with Tk-ChiA and Tk-GlmA (Fig. 1B). Bacterial Strains, Plasmids, and Media—T. kodakaraensis KOD1 was grown anaerobically for 48 h at 85 °C in 1-liter screw-cap bottles with 800 ml of MA medium (4.8 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 pyruvic acid sodium salt (5 g/liter) and chitin (1 g/liter). Escherichia coli TG-1 and BL21-CodonPlus(DE3)-RIL were used as hosts for the expression plasmid derived from pET-15b (Novagen, Madison, WI) and were cultivated in LB medium at 37 °C. Partial Purification of GlcNAc2 Deacetylase from T. kodakaraensis KOD1—The culture broth of T. kodakaraensis containing chitin (11.2 liters) was filtered through Toyo filter paper No. 101 (Toyo Roshi, Tokyo, Japan) to obtain chitin-associated cells. The cells on the chitin particles were suspended in buffer A (50 mm Tris-HCl (pH 9.0), 1 mm EDTA) and then disrupted by sonication. The supernatant obtained by centrifugation (6000 × g for 15 min at 4 °C) was subjected to ammonium sulfate fractionation, and the 50–70% ammonium sulfate-precipitated fraction was dissolved in 45 ml of buffer A. The protein solution was applied to an anion-exchange Resource Q column (6 ml) (Amersham Biosciences) equilibrated with buffer A containing 0.2 m NaCl and eluted with a linear gradient of 0.2–0.5 m NaCl. Fractions containing GlcNAc2 deacetylase activity were collected and concentrated using Ultrafree-4 centrifugal filter unit Biomax-30 (Millipore, Bedford, MA). The sample was then applied to a gel-filtration Superdex-200 HR 10/30 column (Amersham Biosciences) equilibrated with buffer B (50 mm Tris-HCl (pH 8.0), 150 mm NaCl). The collected active fractions were dialyzed against buffer C (20 mm Tris-HCl (pH 7.5)) and applied to an anion-exchange Mono Q HR 5/5 column (Amersham Biosciences). The proteins were eluted with a linear gradient of 0.2–0.5 m NaCl. Ammonium sulfate was added to the active fractions after the Mono Q column at a final concentration of 1.5 m, and the sample was applied onto hydrophobic Resource ISO (1 ml) (Amersham Biosciences) that had been equilibrated with 1.5 m ammonium sulfate in 50 mm Tris-HCl buffer (pH 8.0). The proteins were eluted with a linear gradient of 1.5–0.5 m ammonium sulfate. The active fractions were combined and used for activity staining after polyacrylamide gel electrophoresis. Protein concentration was determined with the Bio-Rad protein assay system with bovine serum albumin as a standard. Activity Staining—GlcNAc2 deacetylase activity was detected in the gel after non-denaturing SDS-PAGE by using the fluorogenic substrate 4-methylumbelliferyl N-acetyl-β-d-glucosaminide (GlcNAc-4MU, Sigma) with recombinant exo-β-d-glucosaminidase from T. kodakaraensis (Tk-GlmA) (9Tanaka T. Fukui T. Atomi H. Imanaka T. J. Bacteriol. 2003; 185: 5175-5181Crossref PubMed Scopus (89) Google Scholar) as a coupling enzyme. A protein sample mixed with 2× sample buffer was applied to 12.5% SDS-polyacrylamide gel without prior boiling. After the electrophoresis, the gel was rinsed three times with 100 mm Tris-HCl (pH 8.0) for 20 min followed by soaking in the reaction buffer (100 μm GlcNAc-4MU, 100 mm Tris-HCl (pH 8.0)) at room temperature for 30 min. The gel removed from the reaction buffer was overlaid with a Tk-GlmA-containing gel (10 nm recombinant Tk-GlmA, 12.5% acrylamide, 0.33% N,N′-methylenebisacrylamide, 100 mm Tris-HCl (pH 8.0), 0.25% ammonium persulfate, 0.05% TEMED), and the fluorescent band was visualized on a transilluminator (312 nm) after incubation of the gel bilayer at 70 °C for 5–15 min. The N-terminal amino acid sequence of the active protein was determined by a protein sequencer Model 491 cLC (Applied Biosystems, Foster City, CA). DNA Manipulations and Sequencing—DNA manipulations were carried out by standard methods, as described by Sambrook and Russell (10Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001Google Scholar). Restriction enzymes and other modifying enzymes were purchased from Takara Shuzo (Kyoto, 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 BigDye Terminator Cycle Sequencing Ready Reaction kit Version 3.0 and Model 3100 capillary DNA sequencer (Applied Biosystems). Construction of the Expression Plasmid—The expression plasmid for Tk-dac was constructed by PCR as described below. Two oligonucleotides (sense, 5′-TCGGCCATGGTGTTTGAGGAGTTCAAC-3′; antisense, 5′-GCGGATCCAGAGAGGTACAG-3′ (underlined sequences indicate an NcoI site in the sense primer and a BamHI site in the antisense primer, respectively)) and T. kodakaraensis genomic DNA were used as primers and template for DNA amplification, respectively. The amplified DNA was digested with NcoI and BamHI and then ligated with the corresponding sites in the plasmid pET-15b. The absence of unintended mutations in the insert was confirmed by DNA sequencing. The resulting plasmid was designated as pET-dac. Purification of Recombinant Tk-Dac—E. coli BL21-CodonPlus(DE3)-RIL cells harboring pET-dac were induced for overexpression with 0.05 mm isopropyl-β-d-thiogalactopyranoside at the mid-exponential growth phase and incubated for a further 3 h at 37 °C. The cells were harvested by centrifugation (5000 × g for 15 min at 4 °C), resuspended in buffer D (50 mm Tris-HCl (pH 7.5), 1 mm EDTA), and then disrupted by sonication. The supernatant after centrifugation (14,000 × g for 30 min) was incubated at 80 °C for 20 min and centrifuged (14,000 × g for 15 min) to obtain a heat-stable protein solution. The solution was applied to ammonium sulfate precipitation at 70% saturation, and the resulting precipitate was dissolved in 1.3 m ammonium sulfate in buffer E (50 mm Tris-HCl (pH 8.0), 1 mm EDTA). Insoluble proteins were removed by centrifugation (28,000 × g for 15 min at 4 °C), and the supernatant was applied to a hydrophobic Resource ISO column (6 ml) (Amersham Biosciences) equilibrated with 0.9 m ammonium sulfate in buffer E. The proteins were eluted with a linear gradient of 0.9–0.45 m ammonium sulfate, and the peak fractions eluting at 0.65 m ammonium sulfate were concentrated using Ultrafree-4 centrifugal filter unit Biomax-10 (Millipore). This was applied to a gel-filtration Superdex-200 HR 10/30 column equilibrated with buffer B. The peak fractions were dialyzed against buffer C and applied to an anion-exchange Mono Q HR 5/5 column. The proteins were eluted with a linear gradient of 0.2–0.5 m NaCl, and the peak fractions were dialyzed with 10 mm Tris-HCl (pH 7.5) and stored at 4 °C. Enzyme Assays—Deacetylase activity during purification of the native enzyme was assayed with a fluorogenic substrate, GlcNAc-4MU, under the presence of excess recombinant Tk-GlmA for coupled hydrolysis. The reaction was performed at 70 °C for 60 min in 500 μl of 10 μm GlcNAc-4MU, 10 nm Tk-GlmA, and 50 mm Tris-HCl (pH 8.0). The reaction was terminated by cooling the sample in an ice-cold bath. To determine the optimal pH for recombinant Tk-Dac, the reaction was performed for 10 min at 70 °C in 250 μl of 10 μm GlcNAc-4MU, 4 nm recombinant Tk-Dac, and 50 mm appropriate buffer (citrate-NaOH, HEPES-NaOH, or CHES-NaOH). After cooling the sample to stop the reaction, the protein was removed from the reaction mixture with a centrifugal filter devise (Microcon YM-10). Removal of the Tk-Dac protein was confirmed by the absence of detectable deacetylation activity in the filtrate. 100 μl of the filtrate was then added into 400 μl of 12.5 nm Tk-GlmA and 50 mm MES-NaOH (pH 6.0). This mixture was incubated at 70 °C for 10 min. This reaction time (10 min) was confirmed to be sufficient to complete the second reaction under the presence of excess Tk-GlmA. In the case of determining optimal temperature, the first reaction was performed in 50 mm HEPES-NaOH (pH 8.5) at various temperatures (37–100 °C). To quantify the liberated 4-methylumbelliferon, the cooled sample (500 μl) was mixed with 500 μl of 100 mm glycine-NaOH (pH 11), and the fluorescence (350 nm, excitation; 440 nm, emission) was measured with a spectrofluorometer (model F-2000; Hitachi, Tokyo, Japan). Kinetic properties of recombinant Tk-Dac toward N-acetylchitooligosaccharides were determined as below. The reactions were performed with various concentrations of GlcNAc1–3 (GlcNAc, 5–160 mm; GlcNAc2, 2.5–80 mm; GlcNAc3, 1.25–40 mm) in 50 mm HEPES-NaOH (pH 8.5) at 75 °C for 5 min. The reactions were stopped by adding 10 μl of 0.5 m HCl at 4 °C, and then Tk-Dac was removed with Microcon YM-10. The amount of acetic acid in the filtrate was enzymatically determined by using an acetic acid determination kit (Roche Diagnostics). Bulk GlcNAc2 and GlcNAc3 were kindly donated from Yaizu Suisankagaku Industry (Shizuoka, Japan). Acetic acid slightly contaminated in these substrates had been removed before use by an anion-exchange spin column (Vivapure Q Mini H; Sartorius, Göttingen, Germany). Analyses of Reaction Products—The analyses of reaction products from GlcNAc1–5, N-acetylgalactosamine, N-acetylmannosamine, N-acetylmuramic acid, and GlcNAc6P were performed with silica gel thin-layer chromatography (TLC) as described previously (5Tanaka T. Fujiwara S. Nishikori S. Fukui T. Takagi M. Imanaka T. Appl. Environ. Microbiol. 1999; 65: 5338-5344Crossref PubMed Google Scholar) except that we used an LHP-KF HPTLC plate (Whatman, Kent, UK) in this study. For detection of the products, aniline diphenylamine reagent and ninhydrin reagent were used. Western Blot Analysis—T. kodakaraensis was cultivated in 10 ml of MA medium supplemented with 20 μl of polysulfide solution (20% elemental sulfur in 3 m Na2S) and various kinds of saccharides. The preparation of colloidal chitin has been described previously (5Tanaka T. Fujiwara S. Nishikori S. Fukui T. Takagi M. Imanaka T. Appl. Environ. Microbiol. 1999; 65: 5338-5344Crossref PubMed Google Scholar). The cells were harvested, disrupted by sonication in buffer C containing protein inhibitor mix (Complete mini; Roche Diagnostics), and then centrifuged (15,000 × g for 30 min) to obtain soluble fractions. Each fraction was subjected to SDS-PAGE and successive Western blot analysis using specific antiserum (rabbit) against the recombinant Tk-Dac. A protein A-peroxidase conjugate was used to visualize the specific protein together with 4-chloro-1-naphthol and hydrogen peroxide. Northern Blot Analysis and Reverse Transcription (RT)-PCR—Cultivation of T. kodakaraensis with GlcNAc2 or colloidal chitin was performed as described above. Total RNA was isolated using the RNeasy Midi kit and the RNase-free DNase Set (Qiagen). For Northern blot analysis, 20 μg of total RNA was separated by denaturing agarose electrophoresis and transferred to a nylon membrane (Hybond-N+; Amersham Biosciences) by capillary blotting. Digoxigenin labeling of a DNA fragment, hybridization, and detection were performed according to the instructions of the manufacturer (DIG High Prime DNA Labeling and Detection Starter Kit I; Roche Diagnostics). A HindIII-NdeI fragment (747 bp) of the Tk-dac-coding region was used as a template for probe preparation. For RT reactions, 250 ng of total RNA was used with Transcriptor reverse transcriptase (Roche Diagnostics), and subsequent PCR was performed with KOD polymerase (for Tk-dac, Tk-glmA, Tk-gly, and Tk-chiA) or KOD dash polymerase (for Tk-sbp) (Toyobo). Sequences of the primer pairs were: F1, 5′-TCGGCCATGGTGTTTGAGGAGTTCAAC-3′, and R1, 5′-GGGCTCCCAAAGCTCCCAC-3′ for Tk-dac; F2, 5′-GTCGCCTGGAGGCACTTCTTC-3′, and R2, 5′-GGGAGTATTGCTCATGGCC-3′ for Tk-glmA; F3, 5′-CGGCCTCTTCGAGGCTGG-3′, and R3, 5′-GTAGAGCTGGACGATGTAC-3′ for Tk-sbp; F4, 5′-CAGAATCTTTCCGTGTCC-3′, and R4, 5′-TCCGTTGCCTTAACGTCC-3′ for Tk-gly; F5, 5′-CGAGACTGCCATAGAGATCC-3′, and R5, 5′-GCAGATCTCAGCCGAGGTGCTGGAGAACAGTATC-3′ for Tk-chiA. Primer pairs were designed to amplify a ∼600 bp fragment of each gene. Immunoprecipitation—Protein A-Sepharose fast flow (Amersham Biosciences) was used as the affinity resin for immunoprecipitation experiments. Five hundred microliters of rabbit antiserum (anti-Tk-Dac serum, anti-Tk-GlmA serum (9Tanaka T. Fukui T. Atomi H. Imanaka T. J. Bacteriol. 2003; 185: 5175-5181Crossref PubMed Scopus (89) Google Scholar), or serum before immunization) was mixed with 100 μl of the resin suspension at 4 °C for 12 h. Antibody-bound resin was centrifuged, and the supernatant was removed. The pellet was washed 5 times with 1.5 ml of phosphate-buffered saline (136.9 mm NaCl, 8.1 mm Na2HPO4·12 H2O, 2.68 mm KCl, 1.47 mm KH2PO4) followed by the addition of 100 μl of the cell extract (2 mg of protein/ml) of T. kodakaraensis grown in the MA medium containing 0.1% GlcNAc2. After incubation at 4 °C for 1.5 h, the supernatant was subjected to determine the GlcNAcase, GlcNase, and deacetylase activities. Identification of the Protein Exhibiting GlcNAc2 Deacetylase Activity and the Corresponding Gene in T. kodakaraensis KOD1—As described above we have previously demonstrated that T. kodakaraensis possesses a chitinase (Tk-ChiA) for degradation of chitin polymer to GlcNAc2 along with a novel exo- β-d-glucosaminidase (Tk-GlmA) to cleave the deacetylated product of GlcNAc2 (5Tanaka T. Fujiwara S. Nishikori S. Fukui T. Takagi M. Imanaka T. Appl. Environ. Microbiol. 1999; 65: 5338-5344Crossref PubMed Google Scholar, 6Tanaka T. Fukui T. Imanaka T. J. Biol. Chem. 2001; 276: 35629-35635Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 9Tanaka T. Fukui T. Atomi H. Imanaka T. J. Bacteriol. 2003; 185: 5175-5181Crossref PubMed Scopus (89) Google Scholar). These results strongly suggested the existence of an enzyme responsible for the deacetylation of GlcNAc2 in this organism. Therefore, the deacetylase activity in T. kodakaraensis was examined by using a fluorogenic Glc-NAc-4MU and recombinant Tk-GlmA as a substrate and a coupling enzyme, respectively. It has been already clarified that Tk-GlmA cannot cleave this substrate due to the N-acetyl group on the sugar moiety nor can it deacetylate this substrate (9Tanaka T. Fukui T. Atomi H. Imanaka T. J. Bacteriol. 2003; 185: 5175-5181Crossref PubMed Scopus (89) Google Scholar). The cell extract of T. kodakaraensis showed only weak cleavage activity (apparent GlcNAcase activity) in the absence of the recombinant Tk-GlmA (4.16 pmol/min/mg). In contrast, the addition of the coupled enzyme enhanced the cleavage activity up to 5.5-fold (23.1 pmol/min/mg), indicating a significant deacetylase activity in the extract. We then performed purification of the protein exhibiting this deacetylase activity from T. kodakaraensis cells. The cells cultivated in chitin-containing medium were disrupted by sonication, and the active protein was partially purified by ammonium sulfate fractionation and column chromatography, as described under "Experimental Procedures." Although many protein bands were still observed in non-denaturing SDS-PAGE after the final chromatography (Fig. 2A, lane 2), a protein with GlcNAc2 deacetylase activity in the gel could be identified by activity staining using GlcNAc-4MU and recombinant Tk-GlmA (Fig. 2A, lane 1, black arrowhead). In the negative control experiment without the coupling enzyme, the deacetylase-active band was not detected (data not shown). The N-terminal amino acid sequence of the identified protein was determined to be VFEEFNNFDEAF. Using the preliminary complete genome sequence of T. kodakaraensis KOD1, we searched for this amino acid sequence and identified one open reading frame that encoded a protein with the N-terminal sequence of MVFEEFNNFDEAFSALL (identical amino acids are underlined). Interestingly, this gene was located adjacently upstream of the Tk-chiA gene in the opposite orientation and also relatively near (∼9 kilobase pairs upstream) Tk-glmA (Fig. 3). This gene was designated as Tk-dac. Primary structure of Tk-dac—The Tk-dac gene consisted of 804 bp, encoding a protein of 268 amino acids with a predicted molecular mass of 30,300 Da. There was neither an N-terminal signal sequence nor transmembrane helices in the deduced amino acid sequence, which was consistent with the fact that Tk-Dac was detected in and partially purified from the cytosolic fraction of T. kodakaraensis cells as an intracellular enzyme. Clear-cut homologs against the translated product were only seen in the closely related Pyrococcus spp., Pyrococcus abyssi (PAB1341), P. furiosus (PF0354), and Pyrococcus horikoshii (PH0499) (60–61% identities), annotated as hypothetical proteins with unknown function. In addition, distantly related but homologous proteins (25–35% identities) are widely distributed in archaea and bacteria. Against the Pfam and COGs databases (11Bateman A. Birney E. Cerruti L. Durbin R. Etwiller L. Eddy S.R. Griffiths-Jones S. Howe K.L. Marshall M. Sonnhammer E.L.L. Nucleic Acids Res. 2002; 30: 276-280Crossref PubMed Scopus (2021) Google Scholar, 12Tatusov R.L. Natale D.A. Garkavtsev I.V. Tatusova T.A. Shankavaram U.T. Rao B.S. Kiryutin B. Galperin M.Y. Fedorova N.D. Koonin E.V. Nucleic Acids Res. 2001; 29: 22-28Crossref PubMed Scopus (1560) Google Scholar), Tk-Dac is classified into the uncharacterized LmbE-like protein family (Pfam02585 as well as COG2120), where LmbE was originally assigned to a hypothetical protein coded in a gene cluster for lincomycin biosynthesis of Streptomyces lincolnensis. This family includes N-acetylglucosaminylphosphatidylinositol de-N-acetylases from mammals (13Nakamura N. Inoue N. Watanabe R. Takahashi M. Takeda J. Stevens V.L. Kinoshita T. J. Biol. Chem. 1997; 272: 15834-15840Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), yeast (14Watanabe R. Ohishi K. Maeda Y. Nakamura N. Kinoshita T. Biochem. J. 1999; 339: 185-192Crossref PubMed Scopus (77) Google Scholar), and protozoa (15Chang T. Milne K.G. Güther M.L.S. Smith T.K. Ferguson M.A.J. J. Biol. Chem. 2002; 277: 50176-50182Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and 1-d-myo-inosityl-2-acetamido-2-deoxy-α-d-glucopyranoside deacetylase from mycobacterium (16Newton G.L. Av-Gay Y. Fahey R.C. J. Bacteriol. 2000; 182: 6958-6963Crossref PubMed Scopus (99) Google Scholar), involved in glycosylphosphatidylinositol anchor biosynthesis and mycothiol biosynthesis, respectively. Although Tk-Dac shared only weak and partial similarities with these enzymes, an important common feature is the function as a deacetylase toward the N-acetylglucosamine moiety. Recently, the crystal structure of a protein with unknown function in the LmbE-like protein family (TT1542) has been determined in the course of structural genomics of Thermus thermophilus (17Handa N. Terada T. Kamewari Y. Hamana H. Tame J.R.H. Park S.Y. Kinoshita K. Ota M. Nakamura H. Kuramitsu S. Shirouzu M. Yokoyama S. Protein Sci. 2003; 12: 1621-1632Crossref PubMed Google Scholar). In the three-dimensional structure, highly conserved residues in this family were clustered in one region with their side chains composing the surface of a hydrophilic cavity. This region was suggested to be a putative active site, and a computational analysis predicted the catalytic importance of seven residues in the cluster. Although the identity between Tk-Dac and TT1542 was 30% within 223 amino acids, 6 of the 7 important residues were also conserved in Tk-Dac (His-40, Asp-42, Asp-43, Arg-88, Glu-91, and Asp-111). Overexpression and Purification of Recombinant Tk-Dac—To characterize Tk-Dac in detail, the recombinant protein was produced in E. coli cells with the pET expression system. The recombinant protein was purified to apparent homogeneity on SDS-PAGE by heat treatment and column chromatography, as shown in Fig. 2B, lane 1. The result of its N-terminal amino acid sequence was identical to the deduced amino acid sequence (MVFEEFNNFDEA, identical amino acids are under-lined). The mobility of the purified protein in the non-denaturing SDS-PAGE (Fig. 2B, lane 2, upper band) corresponded to that of the protein with GlcNAc2 deacetylase activity from T. kodakaraensis in Fig. 2A. The molecular mass of the recombinant Tk-Dac was estimated to be about 30 kDa by denaturing SDS-PAGE and 160 kDa by gel filtration chromatography (figure not shown), and the latter was comparable with that of the active protein from T. kodakaraensis. The results indicated that Tk-Dac was likely to be a homohexameric enzyme. Enzymatic Properties of Tk-Dac—The optimal pH and temperature of Tk-Dac for GlcNAc-4MU were 8.5 and 75 °C, respectively. Activity levels at 37 °C and 100 °C were ∼20% of that observed at the optimal temperature. To examine the mode of action of this enzyme, various chain lengths of N-acetylchitooligosaccharides (GlcNAc1–5) were used as substrates, and the reaction products were analyzed by TLC with two detection procedures. The results are shown in Fig. 4. Tk-Dac could efficiently deacetylate GlcNAc to GlcN. When GlcNAc2–5 were used as substrates, detection of the products with ninhydrin reagent (for amino sugars) (Fig. 4A) clearly indicated formation of deacetylated products from all the substrates examined. However, the mobilities of each reaction product did
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