Isolation of a Glucosamine-specific Kinase, a Unique Enzyme ofVibrio cholerae

We showed previously that chitin catabolism by the marine bacterium Vibrio furnissii involves at least three signal transduction systems and many genes, several of which were molecularly cloned, and the corresponding proteins were characterized. The predicted amino acid sequences of these proteins showed a high degree of identity to the corresponding proteins from Vibrio cholerae, whose complete genomic sequence has recently been determined. We have therefore initiated studies with V. cholerae. We report here a novel ATP-dependent glucosamine kinase of V. cholerae encoded by a gene designated gspK. The protein, GspK (31.6 kDa), was purified to apparent homogeneity from recombinant Escherichia coli. The product of the reaction was shown to be GlcN-6-P by matrix-assisted laser desorption/ionization-time of flight (MALDI mass spectrometry) and NMR. The Km values for GlcN, ATP, and MgCl2 were 0.45, 2.4, and 2.2 mm, respectively, and the Vmax values were in the range 180–200 nmol/μg/min (∼6 nmol/pmol/min). Kinase activity was not observed with any other sugar, including: galactosamine, mannosamine, Glc, GlcNAc, GalNAc, mannose, 2-deoxyglucose, and oligosaccharides of chitosan. The enzyme is also ATP-specific. The kinase can be used to specifically determine micro quantities of GlcN in acid hydrolysates of glycoconjugates. The physiological function of this enzyme remains to be determined. We showed previously that chitin catabolism by the marine bacterium Vibrio furnissii involves at least three signal transduction systems and many genes, several of which were molecularly cloned, and the corresponding proteins were characterized. The predicted amino acid sequences of these proteins showed a high degree of identity to the corresponding proteins from Vibrio cholerae, whose complete genomic sequence has recently been determined. We have therefore initiated studies with V. cholerae. We report here a novel ATP-dependent glucosamine kinase of V. cholerae encoded by a gene designated gspK. The protein, GspK (31.6 kDa), was purified to apparent homogeneity from recombinant Escherichia coli. The product of the reaction was shown to be GlcN-6-P by matrix-assisted laser desorption/ionization-time of flight (MALDI mass spectrometry) and NMR. The Km values for GlcN, ATP, and MgCl2 were 0.45, 2.4, and 2.2 mm, respectively, and the Vmax values were in the range 180–200 nmol/μg/min (∼6 nmol/pmol/min). Kinase activity was not observed with any other sugar, including: galactosamine, mannosamine, Glc, GlcNAc, GalNAc, mannose, 2-deoxyglucose, and oligosaccharides of chitosan. The enzyme is also ATP-specific. The kinase can be used to specifically determine micro quantities of GlcN in acid hydrolysates of glycoconjugates. The physiological function of this enzyme remains to be determined. phosphoenolpyruvate matrix-assisted laser desorption/ionization-time of flight {[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid We have reported that the chitin catabolism cascade in marine bacterium Vibrio furnissii comprises several signal transduction pathways and many proteins (reviewed in Ref. 1Keyhani N.O. Roseman S. Biochim. Biophys. Acta. 1999; 1473: 108-122Crossref PubMed Scopus (250) Google Scholar). Among the family Vibrionaceae, Vibrio cholerae is one of the most important with respect to human health and disease. It is a Gram-negative marine bacterium and a human intestinal pathogen that resides in both brackish water and seawater. In the marine environment,V. cholerae is closely associated with copepods, microscopic crustacea that comprise the most abundant animals on earth. Seasonal blooms in the copepod population in inhabited regions, such as the Ganges delta, coincide with blooms in the chitinivorous V. cholerae and outbreaks of cholera in the human residents (2Nalin D.R. Lancet. 1976; 2: 958Abstract PubMed Scopus (36) Google Scholar). The bacteria are said to be protected against stomach acids, the major barrier against human infection, by “burrowing” into the copepod cuticles (3Nalin D.R. Daya V. Reid A. Levine M.M. Cisneros L. Infect. Immun. 1979; 25: 768-770Crossref PubMed Google Scholar). Although we have acquired a significant body of information on the chitin catabolic cascade in the bacterium V. furnissii, little is known concerning the pathway in V. cholerae. The complete DNA sequence of the V. cholerae genome has recently been reported (4Heidelberg J.F. Eisen J.A. Nelson W.C. Clayton R.A. Gwinn M.L. Dodson R.J. Haft D.H. Hickey E.K. Peterson J.D. Umayam L. Gill S.R. Nelson K.E. Read T.D. Tettelin H. Richardson D. Ermolaeva M.D. Vamathevan J. Bass S. Qin H. Dragoi I. Sellers P. McDonald L. Utterback T. Fleishmann R.D. Nierman W.C. White O. Nature. 2000; 406: 477-483Crossref PubMed Scopus (1447) Google Scholar) and, as we shall show elsewhere, there is a high degree of identity in the predicted amino acid sequences of the proteins we have identified in V. furnissii and their presumptive counterparts in V. cholerae. A number of genes identified previously in V. furnissii appeared to be clustered in the genome of V. cholerae, and since they may constitute a chitin degradation operon, we are currently attempting to identify the functions of the other presumptive genes by subcloning them into Escherichia coli, expressing the proteins, and determining their functions. As shown under “Experimental Procedures,” we have cloned a gene, designated gspK (0.8 kb), encoding a glucosamine-specific kinase, GspK (31.6 kDa), from the chromosomal DNA of V. cholerae. Furthermore, analysis of the nucleotide sequence ofgspK and its putative amino acid sequence indicated that the predicted amino acid sequences of GspK from V. cholerae are 100% identical to a hitherto unrecognized gene in V. furnissii. Although the ubiquitous enzyme, hexokinase, phosphorylates many sugars, including glucosamine and mannosamine (5Jourdian G.W. Roseman S. Biochemical Preparations. 1962; 9: 44-47Google Scholar), and although a GlcNAc-specific ATP-dependent kinase (6Asensio C. Ruiz-Amil M. Methods Enzymol. 1966; 9: 421-425Crossref Scopus (21) Google Scholar) is present in V. furnissii (7Yu B.L. Bassler C. Lee Y.C. Roseman S. J. Biol. Chem. 1991; 266: 24276-24286Abstract Full Text PDF PubMed Google Scholar), there are apparently no previous reports on a GlcN-specific ATP-dependent kinase. 1An extract of Schistosoma mansoni(28Bueding E. Ruppender H. Mackinnon J. Proc. Natl. Acad. Sci. U. S. A. 1954; 40: 773-777Crossref PubMed Google Scholar), known to contain several sugar kinases, was found to phosphorylate GlcN, as well as other sugars such as Glc. It was partially fractionated, and the kinase activity for GlcN was separated from that for Glc. However, no other sugars were assayed with the GlcN active fraction. The following chemicals, reagents, and materials were purchased from the indicated sources: ADP, ATP, P-enolpyruvate,2 NADH, Glc, Fru, Man, Gal, GlcN, GlcNAc, mannosamine (ManN), galactosamine (GalN), and other carbohydrates from Sigma unless otherwise indicated; chitosan oligosaccharides (GlcN)n, n = 1–3, and chitin oligosaccharides (GlcNAc)n, n = 1–2, from Seikagaku America, Inc. (Rockville, MD); glucose oligosaccharides (Glc)n, n = 1–5 and reagents for bacterial media from Difco and J. T. Baker. Reagents for molecular biology were obtained from New England Biolabs (Beverly, MA), Stratagene, and U. S. Biochemical Corp. Pyruvate kinase and lactic acid dehydrogenase for the kinase assay were purchased from Sigma. Other buffers and reagents were of the highest purity commercially available. V. cholerae E1 Tor N16961 is the strain used for obtaining the DNA sequences of the two chromosomes (4Heidelberg J.F. Eisen J.A. Nelson W.C. Clayton R.A. Gwinn M.L. Dodson R.J. Haft D.H. Hickey E.K. Peterson J.D. Umayam L. Gill S.R. Nelson K.E. Read T.D. Tettelin H. Richardson D. Ermolaeva M.D. Vamathevan J. Bass S. Qin H. Dragoi I. Sellers P. McDonald L. Utterback T. Fleishmann R.D. Nierman W.C. White O. Nature. 2000; 406: 477-483Crossref PubMed Scopus (1447) Google Scholar) and was used in the present work, along with V. furnissii 1519, which is similar to 1514, the strain used in our earlier work. BothVibrios were grown either in high salt LB (LMB, Luria broth supplemented with an additional 10 g of NaCl/liter) or in minimal media 50 mm HEPES buffer, pH 7.5, containing 50% artificial seawater, 0.2% NH4Cl, 0.01% K2HPO4, and 0.5% dl-lactate (7Yu B.L. Bassler C. Lee Y.C. Roseman S. J. Biol. Chem. 1991; 266: 24276-24286Abstract Full Text PDF PubMed Google Scholar).E. coli strains BL21(DE3) (Novagen) and XL1-Blue (Stratagene) harboring designated plasmid constructs were stored as frozen cultures in LB with 5% glycerol at −80 °C. Typically,E. coli strains were grown overnight in LB media plus appropriate antibiotics with vigorous shaking. Fresh medium was inoculated with cells from the overnight culture at a 1:20 dilution, and this culture was grown to mid-exponential phase, usually to a density of OD660 = 0.3–0.4 at 37 °C with vigorous aeration. DNA preparations, restriction enzyme digests, ligation, and transformations were performed using standard techniques (8Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 1–3. John Wiley & Sons, Inc., New York1996Google Scholar). The glucosamine-specific kinase gene, gspK, located at VC0614 in the V. cholerae genome was amplified by PCR using synthesized primers based on the gene sequence of the chromosomal DNA of V. cholerae. The 5′ PCR primer was designed to contain anNdeI restriction site to facilitate cloning into the start site following a T7 promoter in the overexpression vector pET21a (Novagen, Madison, WI). The primers used to construct the overexpression plasmid were: 5′-AGAAAACATATGCTGTTACTGACCAA-3′ and 5′-GCAAAGCTTATTCGATAACTCAT-3′. The amplified PCR fragment (2.5 kb) contained both the bglA gene (β-1,4-d-glucosidase, 1.7 kb) to be described elsewhere and the gspK gene (0.8 kb). The PCR fragment was transferred into the NdeI (5′ end) and HindIII (3′ end) of pET21a. The 2.5-kb PCR fragment contains BamHI andHindIII restriction enzyme sites, between which is the DNA sequence for the entire gspK (0.8 kb) gene. The fragment was isolated, ligated into pET21a, and transformed into the T7 polymerase-inducible host strain BL21(DE3). Transcription in BL21(DE3) is under control of the T7 promoter. The translation start site for the gene is 79 bp downstream from the BamHI restriction site. Control cells consisted of wild type E. coli and of the same strain transformed with the vector pET21a without the DNA insert. Extracts of the controls exhibited no GspK activity. Protein concentration was measured with the Bio-Rad protein assay using bovine serum albumin as the standard. A single colony of E. coli strain BL21(DE3) harboring pET:gspK was inoculated into 100 ml of LB medium supplemented with 50 μg/ml ampicillin and grown overnight at 37 °C with vigorous shaking. Two liters of LB medium supplemented with 1 mmisopropyl-1-thio-β-d-galactopyranoside (final concentration) in a 6-liter flask was inoculated with 50 ml of the overnight culture and allowed to grow at 37 °C with aeration until OD600 = 0.3–0.4. The cells were harvested by centrifugation at 4,000 rpm for 30 min at 4 °C. Subsequent steps were performed at 4 °C unless otherwise specified. The cell pellet (4.1 g) was washed twice with 800 ml of 20 mm Tris chloride buffer containing and 1 mm pH 7.5, and in ml of 20 mm Tris chloride buffer with 1 mm pH The cells were by two a cells were by centrifugation at g for 1 were using of of with The was for an additional 1 and at g for 30 in the were by the of to a concentration of The was for an additional 1 and at g for 1 The pellet was in 30 ml of 20 mm buffer containing 50 pH and against the same The was transferred to a with the buffer in the was the was washed with of buffer, and a from 50 mm to in the 20 mm buffer was The activity between and the active were and against 20 chloride buffer, pH 7.5, containing The from 4 was transferred to an which was with The was washed with 10 of with 20 mm Tris chloride buffer, pH 7.5, containing The was and the was washed with of the same buffer and with a from to in the same The active were and against 20 mm Tris chloride buffer, pH 7.5, containing 50 mm and 1 mm and against the same buffer containing 1 mm without The active from were transferred to a which been with 20 mm Tris chloride buffer, pH 7.5, containing 50 mm the was washed with of the same buffer, and a from 50 mm to was to the The activity between and The active were and against 20 mm Tris chloride buffer, 10 mm pH was the by The purified was stored in with mm concentration) at or −80 °C until used for In the of were have been used in this for the of synthesized by and ATP, by the the of or or The is separated from the by or or In the present we to by a assay of the and of a 100 for our by of this type of assay has been used for sugar kinase In the GlcN, ATP, and were with the enzyme, and the reaction was by In the the of by the kinase was with kinase, NADH, and GlcN was from controls to for any from such as Protein to be assayed were to the following (final 100 mm Tris chloride buffer, pH 10 mm 10 mm GlcN, or other sugar The enzyme reaction was initiated by the of the protein to be assayed or of of the purified enzyme otherwise were at or 37 on the for apparently were were overnight at °C. were by for min at 100 °C. The of in the kinase reaction was measured by determining either the of in the assay or the of which was found to be to was at 37 °C in cell a from was to 1 ml containing the following mm Tris chloride buffer, pH mm mm mm The reaction was by of the kinase and lactic acid and the was measured for min or until no was The at was in was observed with enzyme as the control or by of either of the In the and the of was measured by determining the of the the in as a function of Although this function is the of the complete the of is and a to the was used to determine the of these The were found to be to the of in the reaction in the range of was also to and the of protein used in the of the assay and was the of in the of glucosamine was to in the complete kinase reaction were the kinase 5% of the protein is The amino acid of purified recombinant enzyme glucosamine-specific kinase GspK were by using an protein of The of were with the purified enzyme at 37 °C to determine the for a concentration of mm was The following buffer systems were used for the pH buffer, pH buffer, pH Tris chloride buffer, pH buffer, pH and buffer, pH pH were used with The of on enzyme activity was using mm Tris chloride buffer, pH supplemented with or Two of studies were The of the were between 4 and °C. The of the enzyme was measured the range from 4 to °C using the purified enzyme in of mm Tris buffer, pH at the for 30 to and by the activity was as described was purified by as described (5Jourdian G.W. Roseman S. Biochemical Preparations. 1962; 9: 44-47Google Scholar). the following materials are in 100 ml of and MgCl2 The was to pH with and the reaction was initiated by of purified kinase. The pH was at the at 37 by the of and was by to 100 °C for protein was by and the was to pH with acid and a containing ml of 50 The major ADP, and the the glucosamine was The was with and were and of purified sugar were by matrix-assisted mass in both the and by of The were obtained with a with the in We have reported cloning a gene N.O. Roseman S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and gene N.O. Roseman S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Google Scholar) from V. furnissii. are in the chitin degradation analysis of the cloned from V. furnissii that there were two putative (0.8 and 1.7 kb) of the gene. many of the chitin catabolic genes that we have cloned from V. furnissii are also present in the genome of V. cholerae, studies on the two designated kb) and bglA kb), were with V. cholerae. The of bglA be described In the studies reported was molecularly cloned from V. Tor N16961 into pET21a, and the protein was and The enzyme was purified from of E. coli BL21(DE3) harboring pET:gspK as described under “Experimental The product was an apparently protein which at to the The sequence of the sequence is identical to the amino acid sequence predicted from the nucleotide The DNA sequence of the gene that the protein, is and contains amino of recombinant glucosamine-specific of enzyme 1 under the following mm Tris chloride buffer, pH 10 mm ATP, 10 mm and mm activity The purified protein exhibited 50% of the activity obtained in of enzyme 1 under the following mm Tris chloride buffer, pH 10 mm ATP, 10 mm and mm activity The purified protein exhibited 50% of the activity obtained in in a activity was as a function of and other as described under “Experimental In the following shown in were that product was to protein concentration and of The pH of the purified enzyme was between pH the pH enzyme activity was measured as a function of with and was a of on the enzyme, the activity at salt GspK a from °C. However, the enzyme is not to for in the range and at least of its activity at 37 °C. 100% of its activity. It be that these studies were at enzyme and activity may have been at or in the of such as albumin and the enzyme The of concentration on activity were the concentration of ATP, and The of and and and glucosamine were from Biochemical John Wiley & Sons, Inc., New Scholar), and were found to Km = = ATP, Km = = = The are based on the of The enzyme was for ATP, activity with and no activity with or GspK is for The following sugars were in for as as galactosamine, mannosamine, 2-deoxyglucose, mannose, the and the It appeared that the enzyme be used for the of that the of in the assay was to the of GlcN to the in the range using standard assay The most in hydrolysates of is that many of these contain both GlcN and was therefore at to the and was found to have no as a the activity of the enzyme GlcN not was important to determine GlcN be in hydrolysates contain many of amino and these with the for Two of containing were therefore with 4 for at 100 the acid was in and the were and into of was by and the other was by the kinase both the same not The hydrolysates and were performed by and C. Lee of this that the kinase can be used to specifically GlcN in of sugars, amino acids, Two were used to the product from the and NMR. The of the acid J. Roseman S. J. Biol. Chem. Full Text PDF PubMed Google is a the molecular mass of the acid is was used in the a of was as well as an additional at In the the major was at and there were two one at and one at The molecular mass are with the and of The of the in the product was identified by a and by with standard GlcN-6-P J. Roseman S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). 4 the and that the are The following values and were obtained = = = = = = = = = = = = = of show that the is to of showed the as the standard in the was a of and that was not signal for the at is in a at this The of a glucosamine kinase are described in this to the a of with the and the of GlcN from the proteins was with an The standard assay used in this report can determine 10 of GlcN are for the in the in which the is comprises a of and in the in which the is the is In the standard 10 of 100 of the are to the and this can be of the be or which a in The of a glucosamine-specific kinase in V. cholerae to an important function The enzyme is is the of GlcN in the the (GlcNAc)n, the a N.O. Roseman S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) designated 1 in the The of and them to the cell in the In the are by two a and a N.O. Roseman S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google N.O. Roseman S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), to two and The GlcNAc, is by the S. Roseman S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google PubMed Google Scholar), specifically by The gene has been cloned from V. furnissii, and the protein has been Roseman S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The reaction of the The of involves two a and a S. J. Biol. Chem. Full Text PDF PubMed Google Roseman S. J. Biol. Chem. Full Text PDF PubMed Google Roseman S. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and The of E. coli, containing the and genes, has been by J. PubMed Scopus Google J. 1991; PubMed Scopus Google J. PubMed Scopus Google Scholar). are two other for the In V. furnissii, both the cell by and in the from oligosaccharides is by a N.O. Lee Y.C. Roseman S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) In the the is by a N.O. Roseman S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Google Scholar), and We that the is to the by a known to in J. Biol. Chem. Full Text PDF PubMed Google Scholar) and other cell A. J. Biol. Chem. Full Text PDF PubMed Google Methods Enzymol. 1966; Scopus Google Scholar). The of is a GlcNAc-specific ATP-dependent kinase (6Asensio C. Ruiz-Amil M. Methods Enzymol. 1966; 9: 421-425Crossref Scopus (21) Google Scholar) that is found in V. furnissii (7Yu B.L. Bassler C. Lee Y.C. Roseman S. J. Biol. Chem. 1991; 266: 24276-24286Abstract Full Text PDF PubMed Google Scholar). The from the by the is to by this kinase, The reaction shown in is the glucosamine kinase described in this However, in the known pathway in the there is no for the is its GlcN or GlcN, or is GlcN in the GlcN is there are at least two chitin is not contains from 10 to amino GlcN the and described in or similar are active with such as The for the is the E. coli enzyme is also active with S. J. Biol. Chem. Full Text PDF PubMed Google Scholar), which However, coli enzyme an high Km for this the V. cholerae may be active with for by the determine GlcN can be wild cholerae, V. furnissii, and E. were on with GlcN, GlcNAc, and Glc. The three cell of the In a of the three strains were in containing one of the of the strains well on of the three sugars as the of were performed by GlcN is the cell our no such has been In many and that GlcN, the is the PubMed Google Scholar), which the for major pathway for the of GlcN by the in E. coli is the of proteins, which is is shown in the in has been cloned and from V. furnissii and is the of E. coli Roseman S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). V. furnissii not phosphorylate is to be with of the present studies is to determine GlcN the V. cholerae In the of this V. cholerae kinase to be or the kinase function in this is the of