Vol.
174,
January
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
31, 1991
RESEARCH
COMMUNICATIONS Pages
846-852
HIGH LEVEL EXPRESSION AND PURIFICATION OF dTHYMIDINE DIPHOSPHO-D-GLUCOSE 4,6-DEHYDRATASE (yfbB) FROM SALMONEU SEROVAR TYPHIMURIUM LT2 Lajwant K. Romana *, Fernando S. Santiago and Peter R. Reeves Department of Microbiology, The University of Sydney, Sydney, N.S.W. 2006 Australia Received
December
19,
1990
The rfbB gene (dThymidine-diphospho-D-glucose-4,6-dehydratase) from Salmonella serovar typhimurium LT2 was cloned and over-expressed using the T7 RNA polymerase/promoter system. The expressed protein, which represents almost 10% of the total cellular protein was purified 14fold. dTDP-D-glucose 4,6-dehydratase is a homodimer of 43 kDa subunits, is highly specific for dTDP-D-glucose and shows a Km of 427 pM and Vmax of 0.93 pmoles min-1 pg-i of protein for dTDP-D-glucose. The N-terminal analysis confirmed the start position of the gene in the DNA sequence. Complete deactivation of the enzyme by the addition of pchloromercurisulfonic acid and total reactivation by the addition of mercaptoethanol, co-factor NAD’ and cystein showed that a -SH group of the cysteine is involved in the catalytic site. 0 1991Rcademlc PreSSI Inc. The biosynthesis and assembly of O-antigen is encoded by the rfb gene cluster at position 42 min of the genetic map of Salmonella [l]. The O-antigen being polymorphic, provides the basis for a major serological typing scheme. The cloning of a part of this cluster [2] and the complete physical map [3] have been established. Identification of some genes and the complete structure and sequence of the gene cluster were then determined [4-61. In group B Salmonella serovar typhimurium, the O-antigen is a repeat unit of four hexoses: namely, rhamnose, abequose, mannose and galactose. rfbB is the second gene in the rhamnose synthesis pathway, the first being the rfbA gene responsible for the conversion of glucose-l-phosphate to dTDP-D-glucose. rfbB gene encodes dTDP-D-glucose 4,6-dehydratase (E.C. 4.2.1.46) which * To whom correspondence should be addressed. Abbreviations: EDTA, ethylenediaminetetraacetic acid; SDS-PAGE, Sodium Dodecyl-Sulfate-Polyacrylamide Gel Electrophoresis; FPLC, Fast Protein Liquid Chromatography.
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catalyzes the conversion of dTDP-D-glucose to dTDP-4-keto-6-deoxy-Dglucose. Escherichia coli K12 also synthesise rhamnose and the enzyme dTDPD-glucose has been studied. The mechanism of reaction was established by Melo et al. [7] and Gabriel and Linquist [8]. The over-all reaction is the hydrogen from C-4 of glucose is transferred intramolecularly to C-6 of 4keto-6-deoxyglucose, and the hydrogen at C-S of 4-keto-6-deoxyglucose is derived from the medium. Several studies on isolation, purification and physico-chemical analysis have been done on this enzyme from E.coli 19-121. The same enzyme was also purified from Pseudomonas aeruginosa [13], Pasteurella pseudotuberculosis [ 141, other antibiotic-producing bacteria [ 1517] as well as from plant [ 181 and mammalian [19] sources. In the only report of the enzyme from Salmonella LT2 strain [20], two distinct peaks of activity were observed on DEAE-cellulose column chromatography of the crude extracts which disappeared in the deletion mutants. This was attributed to the presence of two structural genes or the random association of two nonidentical subunits. No further purification or characterization of the enzyme was undertaken. Futhermore, the DNA sequence of the whole rfb gene cluster carried out in our laboratory [6] revealed three possible start positions for the rfbB gene. In this paper, we report the high level expression and isolation of dTDP-D-glucose 4,6-dehydratase from Salmonella serovar typhimurium (LT2) alongwith the N-terminal amino acid sequence and compare the physico-chemical properties of the purified enzyme to those of the E.coli enzyme. MATERIALS
AND
METHODS
Strains and Plasmids E.coli K12 strain HMS174 and expression vectors, pT7-5 and pGPl-2 [21] were kindly provided by Dr. Stanley Tabor (Department of Biological Chemistry, Harvard Medical School, Boston, Mass.). pPRlOl0 is a pUC9 plasmid with a ml 11 fragment containing rfbB DNA [6]. Enzymes and Chemicals Restriction enzymes, DNA polymerase I and T4 DNA ligase were purchased from Boehringer Mannheim. [35S]Methionine was obtained from Amersham. All the nucleotide substrates for the enzyme specificity study were from Sigma Chemical Co. All chemicals used were of analytical grade. Enzyme Assay dTDP-D-glucose 4,6-dehydratase was assayed according to Zarkowsky and Glaser [l I]. One unit of activity is defined as the amount of enzyme that catalyzes the formation of 1 umole of product per min at 370 C. 847
Vol.
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No.
2, 1991
BIOCHEMICAL
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COMMUNICATIONS
Purification of dTDP-D-Glucose 4,6-dehydratase Plasmid pPRlOl0 was digested with EcoRl and Hind111 and the fragment was cloned into the same sites of the polylinker of the expression vector pT7-5 to give pPRl162 and transformed into strain HMS174. Plasmid pGPl-2 containing theT7 polymerase gene was co-transformed into the same strain. Induction of gene expression was carried out as described [21]. Harvested cells were washed once and suspended in 50 mM Tris-HCl, 10 mM MgC12, 1 mM EDTA, pH 7.5. After disrupting the cells in a French Press, the cell debris was removed by centrifugation at 15000 rpm for 10 min. The supematant was treated with streptomycin sulfate to a final concentration of 0.8%. After removing the precipitate, the supernatant was dialyzed against 10 mM Tris-HCl, pH 7.5. The dialysed enzyme which represented 81% of the total activity was applied to a preparative column XK 24 packed with Fast flow Q-Sepharose (Pharmacia, LKB) using a superloop (Pharmacia, LKB) and eluted with a salt gradient from O-l M NaCl in 10 mM Tris-HCl, pH 7.5. This was followed by gel filtration using Superose 12 column (Pharmcia, LKB) with 50 mM Tris-HCl, pH 7.5. The column was calibrated using the low molecular weight standards from Pharmacia (cat no.17-0446-01). To separate the enzyme from a similar size contaminant the eluent was finally subjected to Mono Q ion exchange column chromatography (Pharmacia,LKB) under the same conditions as described for XK 24 column. The column chromatography was performed using a Pharmacia FPLC system. All isolation and purification steps were done at 40 C except for FPLC which was done at room temperature. Other Methods Polyacrylamide gel electrophoresis, fixing, staining and destaining of gels was as described by Maniatis et al. [22]. Exclusive labelling of expressed protein using [35S]methionine was done according to Tabor and Richardson [21]. Protein was estimated using the method of Lowry et al. [23]. N-terminal analysis was carried out by the CSIRO Division of Food Processing, Sydney, Australia. RESULTS
AND DISCUSSION
When cultures of E. coli HMS174 containing rfbB were temperatureinduced, dTDP-D-glucose 4,6-dehydratase accumulated over a 2 h period. The over-expressed protein represented almost 10% of the total cellular protein as determined by densitometry of Commassie-stained gels (Fig. la, note that commassie blue staining gives only a qualitative estimate), and [35S]methionine labelling revealed a single band of expressed protein at 43 kDa on SDS-PAGE (Fig. lb). A 14-fold purification of the enzyme from the crude extract was achieved by the purification scheme presented in Table 1. ‘The N-terminal analysis of the purified protein revealed the start position of rfbB as Met-Lys-Be-Leu-Ile-Thr-Gly-Gly-Ala-Gly. Based on this start position and the DNA sequence [6], the calculated molecular weight of the protein is 40,686 daltons. Gel filtration revealed a single peak with a 848
Vol.
174,
No.
2, 1991
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B
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Figure 1. Electrophoretic pattern of rfbB A. O.l%SDS-14%PAGE of lane 1, uninduced crude extracts of pPRl162; lane 2, induced crude extracts of pPRl162. B. Autoradiograph of [35S]methionine-labelled cells. Cells were pulsed as described [21] and crude extracts were applied on O.l%SDS-1 l%PAGE. Autoradiography was done overnight at -700 C; lane 1, uninduced vector pT7-5; lane 2, induced vector pT7-5; lane 3, uninduced pPR1162; lane 4, induced pPR1162. Protein size markers (in kDa) are denoted on the side. weight of 77 kDa and non denaturing PAGE gave a band at 80 kDa whereas on a denaturing PAGE, the enzyme has an estimated molecular weight of 43 kDa. These data indicate that rfbB is a dimer of identical subunits as shown previously for the E.coli enzyme [24]. The two peaks of activity obtained by Nikaido et al. [20] can not be explained by the present results. It is possible that the two peaks of activity [20] were represented by monomeric and dimeric proteins, as a single open reading frame
molecular
Table 1. Purification Step
Crude extract XK 24 Superose 12 Mono Q
Total Volume b-4 87.5 9.5 4.5 2.0
of dTDP-D-Glucose 4,6-dehydratase from S.tvDhimurium LT2
Total Protein (w> 1718.5 176.4 58.3 23.2
Total Activity (unit) 3270 1590 950 620
849
Specific Activity (unithg)
1.9 9.0 16.3 26.7
Yield Purification (percent) Factor (fold) 100 49 30 19
1 4.7 8.6 14.1
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Figure 2. Densitometer scan of the purified dTDP-D-Glucose 4,6-dehydratase in O.l%SDS-14%PAGE using LKB 2202 Ultroscan laser densitometer with a chart speed of 5 cm/min.
unambiguously accounted for the rfbB activity in the rfb gene cluster [6]. It is worth noting however, that the ion exchange chromatography resulted in a single peak of activity during purification. The purified enzyme was apparently homogeneous as revealed by gel filtration, PAGE (Fig. 2) and N-terminal analaysis. This preparation was used for the kinetics and other physico-chemical analyses. The optimum pH is 8.5 which is the reported value for the enzyme from E.coli [9,10]. The effect of temperature on the enzyme reaction was also studied and maximum enzyme activity could be obtained at 350 C. A dramatic decrease of about 76% in activity was observed from 450 to 500 C. The Km for dTDP-D-glucose 4,6dehydratase from S.tvphimurium is 4.27 x 10-d M and the calculated Vmax is 0.93 pmole min-1 pg- 1. Zarkowsky et al. [ 121 and Wang and Gabriel [lo] reported much lower Km values: 3.00 x 10-S M and 7.14 x 10-5 M, respectively and Gilbert et al. [9] reported a Vmax of around 7 nmole min-1 pg-1 for the E.coli enzyme. These values indicate that Salmonella has higher intracellular levels of the substrate compared with E.coli. Different nucleotide sugars; namely, ADP-glucose, CDP-glucose, UDP-glucose, GDP-glucose, UDP-galactose, dTDP-mannose, CTP, d?TP and dTDP-glucose, were used as substrate to determine the specificity of the purified enzyme. Among the substrates tested, only dTDP-D-glucose gave enzyme activity. Thus, the enzyme is highly specific for dTDP-D-glucose. Complete deactivation was accomplished by the addition of pchloromercurisulfonic acid (p-cmsa) to a final concentration of 0.01 M (Table 2). When 5-fold molar excess of 2-mercaptoethanol was added over the mercurial, reactivation was not successful. However, after treatment with mercaptoethanol and addition of (0.1 mM) co-factor, NAD+ and cysteine (30 mM), complete reactivation was achieved suggesting a -SH group of cysteine is involved in the catalytic site. The enzyme does not require exogenous NAD+ or NADP+ for activity. Although there is no net oxidation or reduction of NAD+, (data not shown), the enzyme must use bound NAD+ for the reaction 850
Vol.
174,
No.
2, 1991
Table 2. Deactivation
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
and reactivation of dTDP-D-Glucose
Treatment Control Deactivated Deactivated + mercaptoethanol Deactivated + mercaptoethanol Deactivated + mercaptoethanol
COMMUNICATIONS
4,6-dehydratase
Specific Activity (Units&g protein)
+ NAD+ + NAD+ + cystein
1.08 0.00 0.00 0.17 1.02
Deactivation was done by addition of p-CMSA to a final concentration of 10 mM. Reactivation was achieved after addition of 5-molar excess of 2mercaptoethanol, co-factor NAD+ (0.1 mM) and cystein (30 mM). All values given are means of two reactions.
as it can not be reactivated without the addition of NAD+. The E.coli enzyme has shown to contain 1 mole NAD+ per dimer of enzyme [24].
Acknowledgment: We thank the Department University of Sydney for the use of FPLC.
of Biochemistry,
The
REFERENCES [l] Sanderson, K.E. and Roth, J.R. (1989) Microbial. Rev. 52,485-532. [2] Brahmbhatt, H.N., Quigley, N.B. and Reeves, P.R. (1986) Mol. Gen. Genet. 203,172-176. [3] Brahmbhatt, H-N., Wyk, P., Quigley, N.B. and Reeves, P.R. (1988) J. Bacterial. 170, 98-102. [4] Wyk, P. and Reeves, P.R. (1989) J. Bacterial. 171,5687-5693. [5] Vet-ma, N. and Reeves, P.R. (1989) J. Bacterial. 171, 5694-5701. [6] Jiang, X.M., Neal, B., Santiago, F., Lee, S.J., Romana, L.K. and Reeves, P.R. (1990) accepted for publication in Mol. Microbial. [7] Melo, A., Elliot, W.H. and Glaser, L. (1968) J. Biol. Chem. 243, 14671474. [8] Gabriel, 0. and Lindquist, L.C. (1968) J. Biol. Chem. 243, 1479-1484. [9] Gilbert, J.M., Matsuhashi, M. and Strominger, J.L. (1965) J. Biol. Chem. 240, 1305-1308. [lo] Wang, S.F. and Gabriel, 0. (1969) J. Biol. Chem. 244, 3430-3437. [ 1l] Zarkowsky, H. and Glaser, L. (1969) J. Biol. Chem. 244,4750-4756. [12] Zarkowsky, H., Lipkin, E. and Glaser, L. (1970) J. Biol. Chem. 245, 6599-6606. [13] Komfeld, S. and Glaser, L. (1961) J. Biol. Chem. 236, 1791-1799. [14] Gonzalez-Porque, P. and Strominger, J.L. (1977) J. Biol. Chem. 247, 6748-6756. [15] Wahl, H.P. and Grisebach, H. (1979) Biochim. Biophys. Acta. 568,243252. 851
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[16] Matern, H., Brillinger, G.U. and Pape, H. (1973) Arch. Mikrobiol. 88, 37-48. [17] Vara, J.A. and Hutchinson, C.R. (1988) J. Biol. Chem. 263, 1499214995. [18] Liao, T.H. and Baber, G.A. (1972) Biochim. Biophys. Acta. 276, 85-93. [19] Broschat, K.O., Chang, S. and Serif, G. (1985) Eur. J. Biochem. 153, 397-401. [20] Nikaido, H., Levinthal, M., Nikaido, K. and Nakane, K. (1967) Proc. Natl. Acad. Sci. USA 57, 1825-1832. [21] Tabor, S. and Richardson, CC. (1985) Proc. Natl. Acad. Sci. USA 82, 1074-1078. [22] Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press Cold Spring Harbor, New York. [23] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (195 1) J. Biol. Chem. 193, 265-275. [24] Chiknas, S. and Gabriel, 0. (1977) Fed. Proc. 36, 872.
852
174,
January
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
31, 1991
RESEARCH
COMMUNICATIONS Pages
846-852
HIGH LEVEL EXPRESSION AND PURIFICATION OF dTHYMIDINE DIPHOSPHO-D-GLUCOSE 4,6-DEHYDRATASE (yfbB) FROM SALMONEU SEROVAR TYPHIMURIUM LT2 Lajwant K. Romana *, Fernando S. Santiago and Peter R. Reeves Department of Microbiology, The University of Sydney, Sydney, N.S.W. 2006 Australia Received
December
19,
1990
The rfbB gene (dThymidine-diphospho-D-glucose-4,6-dehydratase) from Salmonella serovar typhimurium LT2 was cloned and over-expressed using the T7 RNA polymerase/promoter system. The expressed protein, which represents almost 10% of the total cellular protein was purified 14fold. dTDP-D-glucose 4,6-dehydratase is a homodimer of 43 kDa subunits, is highly specific for dTDP-D-glucose and shows a Km of 427 pM and Vmax of 0.93 pmoles min-1 pg-i of protein for dTDP-D-glucose. The N-terminal analysis confirmed the start position of the gene in the DNA sequence. Complete deactivation of the enzyme by the addition of pchloromercurisulfonic acid and total reactivation by the addition of mercaptoethanol, co-factor NAD’ and cystein showed that a -SH group of the cysteine is involved in the catalytic site. 0 1991Rcademlc PreSSI Inc. The biosynthesis and assembly of O-antigen is encoded by the rfb gene cluster at position 42 min of the genetic map of Salmonella [l]. The O-antigen being polymorphic, provides the basis for a major serological typing scheme. The cloning of a part of this cluster [2] and the complete physical map [3] have been established. Identification of some genes and the complete structure and sequence of the gene cluster were then determined [4-61. In group B Salmonella serovar typhimurium, the O-antigen is a repeat unit of four hexoses: namely, rhamnose, abequose, mannose and galactose. rfbB is the second gene in the rhamnose synthesis pathway, the first being the rfbA gene responsible for the conversion of glucose-l-phosphate to dTDP-D-glucose. rfbB gene encodes dTDP-D-glucose 4,6-dehydratase (E.C. 4.2.1.46) which * To whom correspondence should be addressed. Abbreviations: EDTA, ethylenediaminetetraacetic acid; SDS-PAGE, Sodium Dodecyl-Sulfate-Polyacrylamide Gel Electrophoresis; FPLC, Fast Protein Liquid Chromatography.
Vol.
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No.
2, 1991
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catalyzes the conversion of dTDP-D-glucose to dTDP-4-keto-6-deoxy-Dglucose. Escherichia coli K12 also synthesise rhamnose and the enzyme dTDPD-glucose has been studied. The mechanism of reaction was established by Melo et al. [7] and Gabriel and Linquist [8]. The over-all reaction is the hydrogen from C-4 of glucose is transferred intramolecularly to C-6 of 4keto-6-deoxyglucose, and the hydrogen at C-S of 4-keto-6-deoxyglucose is derived from the medium. Several studies on isolation, purification and physico-chemical analysis have been done on this enzyme from E.coli 19-121. The same enzyme was also purified from Pseudomonas aeruginosa [13], Pasteurella pseudotuberculosis [ 141, other antibiotic-producing bacteria [ 1517] as well as from plant [ 181 and mammalian [19] sources. In the only report of the enzyme from Salmonella LT2 strain [20], two distinct peaks of activity were observed on DEAE-cellulose column chromatography of the crude extracts which disappeared in the deletion mutants. This was attributed to the presence of two structural genes or the random association of two nonidentical subunits. No further purification or characterization of the enzyme was undertaken. Futhermore, the DNA sequence of the whole rfb gene cluster carried out in our laboratory [6] revealed three possible start positions for the rfbB gene. In this paper, we report the high level expression and isolation of dTDP-D-glucose 4,6-dehydratase from Salmonella serovar typhimurium (LT2) alongwith the N-terminal amino acid sequence and compare the physico-chemical properties of the purified enzyme to those of the E.coli enzyme. MATERIALS
AND
METHODS
Strains and Plasmids E.coli K12 strain HMS174 and expression vectors, pT7-5 and pGPl-2 [21] were kindly provided by Dr. Stanley Tabor (Department of Biological Chemistry, Harvard Medical School, Boston, Mass.). pPRlOl0 is a pUC9 plasmid with a ml 11 fragment containing rfbB DNA [6]. Enzymes and Chemicals Restriction enzymes, DNA polymerase I and T4 DNA ligase were purchased from Boehringer Mannheim. [35S]Methionine was obtained from Amersham. All the nucleotide substrates for the enzyme specificity study were from Sigma Chemical Co. All chemicals used were of analytical grade. Enzyme Assay dTDP-D-glucose 4,6-dehydratase was assayed according to Zarkowsky and Glaser [l I]. One unit of activity is defined as the amount of enzyme that catalyzes the formation of 1 umole of product per min at 370 C. 847
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Purification of dTDP-D-Glucose 4,6-dehydratase Plasmid pPRlOl0 was digested with EcoRl and Hind111 and the fragment was cloned into the same sites of the polylinker of the expression vector pT7-5 to give pPRl162 and transformed into strain HMS174. Plasmid pGPl-2 containing theT7 polymerase gene was co-transformed into the same strain. Induction of gene expression was carried out as described [21]. Harvested cells were washed once and suspended in 50 mM Tris-HCl, 10 mM MgC12, 1 mM EDTA, pH 7.5. After disrupting the cells in a French Press, the cell debris was removed by centrifugation at 15000 rpm for 10 min. The supematant was treated with streptomycin sulfate to a final concentration of 0.8%. After removing the precipitate, the supernatant was dialyzed against 10 mM Tris-HCl, pH 7.5. The dialysed enzyme which represented 81% of the total activity was applied to a preparative column XK 24 packed with Fast flow Q-Sepharose (Pharmacia, LKB) using a superloop (Pharmacia, LKB) and eluted with a salt gradient from O-l M NaCl in 10 mM Tris-HCl, pH 7.5. This was followed by gel filtration using Superose 12 column (Pharmcia, LKB) with 50 mM Tris-HCl, pH 7.5. The column was calibrated using the low molecular weight standards from Pharmacia (cat no.17-0446-01). To separate the enzyme from a similar size contaminant the eluent was finally subjected to Mono Q ion exchange column chromatography (Pharmacia,LKB) under the same conditions as described for XK 24 column. The column chromatography was performed using a Pharmacia FPLC system. All isolation and purification steps were done at 40 C except for FPLC which was done at room temperature. Other Methods Polyacrylamide gel electrophoresis, fixing, staining and destaining of gels was as described by Maniatis et al. [22]. Exclusive labelling of expressed protein using [35S]methionine was done according to Tabor and Richardson [21]. Protein was estimated using the method of Lowry et al. [23]. N-terminal analysis was carried out by the CSIRO Division of Food Processing, Sydney, Australia. RESULTS
AND DISCUSSION
When cultures of E. coli HMS174 containing rfbB were temperatureinduced, dTDP-D-glucose 4,6-dehydratase accumulated over a 2 h period. The over-expressed protein represented almost 10% of the total cellular protein as determined by densitometry of Commassie-stained gels (Fig. la, note that commassie blue staining gives only a qualitative estimate), and [35S]methionine labelling revealed a single band of expressed protein at 43 kDa on SDS-PAGE (Fig. lb). A 14-fold purification of the enzyme from the crude extract was achieved by the purification scheme presented in Table 1. ‘The N-terminal analysis of the purified protein revealed the start position of rfbB as Met-Lys-Be-Leu-Ile-Thr-Gly-Gly-Ala-Gly. Based on this start position and the DNA sequence [6], the calculated molecular weight of the protein is 40,686 daltons. Gel filtration revealed a single peak with a 848
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
A
RESEARCH
COMMUNICATIONS
B
12
12
-UL -
3
4
67
Figure 1. Electrophoretic pattern of rfbB A. O.l%SDS-14%PAGE of lane 1, uninduced crude extracts of pPRl162; lane 2, induced crude extracts of pPRl162. B. Autoradiograph of [35S]methionine-labelled cells. Cells were pulsed as described [21] and crude extracts were applied on O.l%SDS-1 l%PAGE. Autoradiography was done overnight at -700 C; lane 1, uninduced vector pT7-5; lane 2, induced vector pT7-5; lane 3, uninduced pPR1162; lane 4, induced pPR1162. Protein size markers (in kDa) are denoted on the side. weight of 77 kDa and non denaturing PAGE gave a band at 80 kDa whereas on a denaturing PAGE, the enzyme has an estimated molecular weight of 43 kDa. These data indicate that rfbB is a dimer of identical subunits as shown previously for the E.coli enzyme [24]. The two peaks of activity obtained by Nikaido et al. [20] can not be explained by the present results. It is possible that the two peaks of activity [20] were represented by monomeric and dimeric proteins, as a single open reading frame
molecular
Table 1. Purification Step
Crude extract XK 24 Superose 12 Mono Q
Total Volume b-4 87.5 9.5 4.5 2.0
of dTDP-D-Glucose 4,6-dehydratase from S.tvDhimurium LT2
Total Protein (w> 1718.5 176.4 58.3 23.2
Total Activity (unit) 3270 1590 950 620
849
Specific Activity (unithg)
1.9 9.0 16.3 26.7
Yield Purification (percent) Factor (fold) 100 49 30 19
1 4.7 8.6 14.1
Vol.
174,
No.
2, 1991
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RESEARCH
COMMUNICATIONS
Figure 2. Densitometer scan of the purified dTDP-D-Glucose 4,6-dehydratase in O.l%SDS-14%PAGE using LKB 2202 Ultroscan laser densitometer with a chart speed of 5 cm/min.
unambiguously accounted for the rfbB activity in the rfb gene cluster [6]. It is worth noting however, that the ion exchange chromatography resulted in a single peak of activity during purification. The purified enzyme was apparently homogeneous as revealed by gel filtration, PAGE (Fig. 2) and N-terminal analaysis. This preparation was used for the kinetics and other physico-chemical analyses. The optimum pH is 8.5 which is the reported value for the enzyme from E.coli [9,10]. The effect of temperature on the enzyme reaction was also studied and maximum enzyme activity could be obtained at 350 C. A dramatic decrease of about 76% in activity was observed from 450 to 500 C. The Km for dTDP-D-glucose 4,6dehydratase from S.tvphimurium is 4.27 x 10-d M and the calculated Vmax is 0.93 pmole min-1 pg- 1. Zarkowsky et al. [ 121 and Wang and Gabriel [lo] reported much lower Km values: 3.00 x 10-S M and 7.14 x 10-5 M, respectively and Gilbert et al. [9] reported a Vmax of around 7 nmole min-1 pg-1 for the E.coli enzyme. These values indicate that Salmonella has higher intracellular levels of the substrate compared with E.coli. Different nucleotide sugars; namely, ADP-glucose, CDP-glucose, UDP-glucose, GDP-glucose, UDP-galactose, dTDP-mannose, CTP, d?TP and dTDP-glucose, were used as substrate to determine the specificity of the purified enzyme. Among the substrates tested, only dTDP-D-glucose gave enzyme activity. Thus, the enzyme is highly specific for dTDP-D-glucose. Complete deactivation was accomplished by the addition of pchloromercurisulfonic acid (p-cmsa) to a final concentration of 0.01 M (Table 2). When 5-fold molar excess of 2-mercaptoethanol was added over the mercurial, reactivation was not successful. However, after treatment with mercaptoethanol and addition of (0.1 mM) co-factor, NAD+ and cysteine (30 mM), complete reactivation was achieved suggesting a -SH group of cysteine is involved in the catalytic site. The enzyme does not require exogenous NAD+ or NADP+ for activity. Although there is no net oxidation or reduction of NAD+, (data not shown), the enzyme must use bound NAD+ for the reaction 850
Vol.
174,
No.
2, 1991
Table 2. Deactivation
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
and reactivation of dTDP-D-Glucose
Treatment Control Deactivated Deactivated + mercaptoethanol Deactivated + mercaptoethanol Deactivated + mercaptoethanol
COMMUNICATIONS
4,6-dehydratase
Specific Activity (Units&g protein)
+ NAD+ + NAD+ + cystein
1.08 0.00 0.00 0.17 1.02
Deactivation was done by addition of p-CMSA to a final concentration of 10 mM. Reactivation was achieved after addition of 5-molar excess of 2mercaptoethanol, co-factor NAD+ (0.1 mM) and cystein (30 mM). All values given are means of two reactions.
as it can not be reactivated without the addition of NAD+. The E.coli enzyme has shown to contain 1 mole NAD+ per dimer of enzyme [24].
Acknowledgment: We thank the Department University of Sydney for the use of FPLC.
of Biochemistry,
The
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