Tm JOURNAL OF BIO~~DGICAL CHEMISTFX 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 1, Issue of January 7, pp. 122-126, 1994 Prinfed in U S A .

Purification, Characterization, and High Performance Liquid Chromatography Assay of SaZrnoneZZa Glucose-1-phosphate Cytidylyltransferase from the Cloned r,%F Gene*

(Received for publication, May 24, 1993, and in revised form, September 1, 1993)

L e ~ a r t LindqvistSg, Rudolf Kaisefl, Peter R. Reevesll, and Alfk Lindberd From the #Department of Clinical Bacteriology, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden, the WePartment of Chemistry I, Karolinska Institute, S-104 01 Stockholm, Sweden, and the [Department of Microbiology, The University of Sydney, Sydney, Australia New South Wales 2006

We report the purification and characterization of glu- cose-1-phosphate cytidylyltransferase, the first of five enzymes committed to biosynthesis of CDP-D-abequose from Salmonella enterica strain LT2. The purification was greatly facilitated by using a cloned rfbF gene en- coding this enzyme. Pure enzyme was obtained by 64- fold enrichment in three chromatography steps. The NH2-terminal sequence of the purified enzyme was in agreement with the sequence predicted from the nucleo- tide sequence of the rfbF gene. The SDS-polyacrylamide gel electrophoresis estimated subunit M, of 31,000 agrees well with the M, of 29,035 calculated from the amino acid composition deduced from the nucleotide sequence of the rfbF gene.

The glucose-1-phosphate cytidylyltransferase cata- lyzes a reversible bimolecular group transfer reaction and steady-state kinetic measurements, including prod- uct inhibition patterns, indicate that this reaction pro- ceeds by a "ping-pong" type of mechanism. The K, val- ues for CTP, a-D-glucose 1-phosphate, CDP-D-glucose, and pyrophosphate are 0.28, 0.64, 0.11, and 1.89 m ~ , re- spectively.

Saccharides, linked to proteins or lipids or occurring as free saccharides, are important surface structures in eukaryotic and prokaryotic cells and are involved in cell recognition phe- nomena. In recent years advances have been made in the chemical synthesis of saccharides (1). However, saccharide syn- thesis is still a relatively complicated procedure, in particular when larger saccharides or large quantities are desirable. In recent years we have become interested in the feasibility of biosynthetic saccharides using in vitro enzymatic synthesis of the Salmonella enterica 0-antigen-specific oligosaccharide and its nucleotide sugar precursors as a model (2).

The 0-antigen part of S. enterica sero-group B strains is a repeating tetramer oligosaccharide composed of abequose, mannose, rhamnose, and galactose assembled from appropri- ate nucleoside diphosphate monosaccharides. The enzymes which participate in the biosynthesis of S. enterica O-antigen- polysaccharide are encoded by genes which are located mostly in the rfb gene cluster (3). The rfb gene cluster has been cloned (4-6) and Escherichia coli K-12 strains, harboring plasmids

* This work was supported by Swedish Medical Research Council Grant 16X-656 and the Australian Research Council. The costs of pub- lication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Bacteriology, Karolinska Inst., Huddinge Hospital, S-141 86 Huddinge, To whom correspondence should be addressed: Dept. of Clinical

Sweden.

containing different parts of the rfb gene cluster of S. enterica LT2, was the source of the enzymes used for in vitro enzymatic synthesis of dTDP-L-rhamnose (2).

The nucleotide sugars which are involved in the biosynthesis of S. entericu lipopolysaccharide, except GDP-D-mannose, all derive from glucose 1-phosphate and a nucleoside triphosphate. The first of five successive steps in the biosynthesis of CDP-D- abequose is the formation of CDP-D-glucose from glucose 1-phosphate and CTP.

CTP + a-glucose 1-phosphate = CDP-D-glucose + pyrophosphate (Eq. 1)

The enzyme which catalyzes the reaction is glucose-l-phos- phate cytidylyltransferase (EC 2.7.7.33), which is encoded by the rfbF gene (7).

Availability of labelled monosaccharide of CDP-D-abequose would facilitate the development of a sensitive assay for esti- mation of abequosyltransferase activity. This would be a useful tool in attempts to localize the abequosyltransferase gene. I t would also facilitate in quantitation for in vitro biosynthesis of the S. enterica tetrasaccharide repeating unit. In this report we describe the purification and properties of the glucose-l-phos- phate cytidylyltransferase using a strain overexpressing the rfbF gene as source of the enzyme.

MATERIALS AND METHODS Chemicals-Inorganic pyrophosphatase (EC 3.6.1.1), dlTP, and CTP

were obtained from Boehringer Mannheim GmbH (Mannheim, Ger- many). ATP, CMP, CDP, GTP, ITP, UTP, ADP-D-glucose, CDP-D-glucose, dTDP-D-glucose, UDP-D-glucose, UDP-D-galactose, UDP-o-mannose, UDP-N-acetyl-D-glucosamine, and all nonradioactive sugar l-phos- phates used were from Sigma. a-~-[U'~C]Glucose 1-phosphate was ob- tained from Du Pont Scandinavia AB (Stockholm, Sweden). The dTDP- L-rhamnose was prepared as described (2).

CDP-[U-14Clglucose was synthesized enzymatically from a-D- [U14Clglucose 1-phosphate and CTP by the purified glucose-l-phos- phate cytidylyltransferase. The incubation mixture contained in 0.05 ml: a-~-[U'~Clglucose 1-phosphate (0.16 pmol, 39.5 pCi), CTP (0.48 pmol), Tris-HC1 buffer, pH 8.0 (3.2 pmol), MgClz (0.8 pmol), 31 milli- units of glucose-1-phosphate cytidylyltransferase and 0.2 unit of inor- ganic pyrophosphatase. The CDP-[14C]glucose formed d e r incubation for 1 h at 37 "C was purified by HPLC' (see below). The yield of the purified CDP-[14C]glucose was 0.12 m o l , and HPLC rechromatography yielded a single peak of absorbance, concomitant with all radioactivity.

Bacterial Strains and Cultivation-E. coli K12 and S. enterica LT2 strains containing different constructs of the Salmonella rfa gene clus- ter from the group B S. enterica strain LT2 were used. The genotypes of the strains are given in Table I. The bacteria were grown aerobically at 37 "C in Luna broth (2% peptone, 1% yeast extract, 0.5% NaCI, and 0.2% glucose, pH 7.4) containing 100 pg of ampicillidml. The cultiva-

The abbreviations used are: HPLC, high performance liquid chro- matography; FPLC, fast protein liquid chromatography; PAGE, poly- acrylamide gel electrophoresis.

122

Glucose-1 -phosphate Cytidylyltransferase of Salmonella 123

tion was camed out under shaking until the culture reached a density of 0.7 a t 530 nm. The cells were then pelleted by centrifugation, washed twice with cold buffer A (50 m Tris-HC1, pH 7.4, 10 m MgC12, and 1 m EDTA), and stored at -20 "C.

Preparation of Crude Enzyme Fractions-The cell paste was thawed in buffer A (0.3 g (wet weight)/ml) and sonicated twice, being frozen in between (five bursts of 15 s each time while being chilled on ice). The cell extract was centrifuged at 30 OOO x g for 30 min at 4 "C. The supernatant fraction was then passed through a Sephadex G-25 column (PD 10, Pharmacia LKB Biotechnology AB, Uppsala, Sweden) using buffer B (50 m Tris-HC1, pH 7.4, I m MgCl, and 22% (v/v) glycerol) as eluate. The protein fraction was collected and used for enzyme purifi- cation or glycerol was added to a final concentration of 50% for storage of the crude enzyme fraction at -20 "C.

Assays-Glucose-1-phosphate cytidylyltransferase activity was measured by following the change of concentration of CTP and CDP- glucose by HPLC analysis. The direction of formation of CDP-glucose from glucose I-phosphate and CTP was used in a standard assay pro- tocol. The reaction mixture (15 pmol of Tris-HC1, pH 8.0, 3.6 pmol of MgC12, 7.2 p o l of glucose 1-phosphate, 1.8 p o l of CTP, 1.8 units of inorganic pyrophosphatase, and an appropriate aliquot of enzyme ( 3 - 100 milliunits) in a total volume of 300 pl) was incubated at 37 "C. Samples (30 p l ) were withdrawn at timed intervals for up to 20 min. The sample was immediately mixed with 1.00 ml of potassium phosphate (50 m, pH 3.0) in order to terminate the reaction. The diluted samples were stored at 4 "C until analysis by HPLC (see below).

From the integrated peak areas of HPLC the amount of CDP-D- glucose formed was calculated as: P X AJAC, where P is the total amount of CMP, CDP and CTP of a blank reaction mixture (without enzyme), calculated from the absorbance at 271 nm assuming molar absorption coefficients of 9.00 m ~ - l cm-'. A, is the integrated area of CDP-D-glucose, and A, is the integrated total area of CMP, CDP, CTP, and CDP-wglucose of the reaction mixture. The initial velocity was expressed as enzyme units defined as micromoles of CDP-D-glucose formed per min.

For determination of kinetic constants the assay was modified. In- organic pyrophosphatase was not included in the reaction mixture. For termination of the enzyme reaction, the sample (30 pl) withdrawn at timed intervals was mixed with only 300 pl of potassium phosphate ( 100 m, pH 3.0) in order to increase the sensitivity of product detection.

The general approach to determining the mechanism by kinetic con- siderations is as described by Cleland (10) and by Segel (11). The ex- periments were designated to provide data by varying concentration of CTP at several fixed concentrations of u-D-glucose I-phosphate or, for both forward and reverse directions, by both substrates varied together while maintaining their concentrations at a constant ratio, [AN = [B] , where A = CTP and B = a-D-glucose 1-phosphate in the forward reaction or A = CDP-D-glucose and B = pyrophosphate in the reverse reaction.

Product inhibition by CDP-o-glucose could not be evaluated without modification. The reaction conditions were as described above, except that [14Clglucose 1-phosphate (2 pCi/pmol) was included. Initial rates were calculated from radiochemical I4C determination of fractionated HPLC effluent and the known specific radioactivity of the [14C]glucose 1-phosphate. The radiochemical determination of CDP-D-glucoSe was used beside the absorbance area determination for all product inhibi- tion experiments.

An unweighted nonlinear last squares approach (Statistica computer program) was used when fitting initial velocity to appropriate equation. The rate equation used for noncompetitive slope or intercept inhibition was,

v = v -[AY(&,A(~ + UYKie) + [AN1 + [IYK,i)Cl+ K,,,RBl)) (Eq. 2)

where A and B represent either CTP or glucose 1-phosphate and [I] the concentration of CDP--glucose or pyrophosphate. Kis and Kii are the constants for the slope and intercept inhibition, respectively. In case of competitive inhibition the term UKii was left out in Equation 2. Values are reported * the standard error of the estimate for each parameter.

Protein was determined by the Coomassie Brilliant Blue G-250 dye- binding method of Bradford (12) using the dye reagent supplied by Bio-Rad and using bovine serum albumin as the standard.

HPLC Analysis-A Supelcosil LC-SAX column (0.46 cm x 25 c m ) equipped with a guard column (LC-SAX supelguard column; Supelco Inc., Bellafonte, PA) was wed for HPLC analysis. Sample (100 pl) or standard solution (0.10 m of nucleotides and nucleotide sugars) was injected onto the column, and the chromatogram was developed with a linear gradient (10 ml) of 0.10-0.40 M potassium phosphate, pH 4.0, followed by 5 ml of 0.10 M potassium phosphate, pH 4.0. The flow rate

was 1.0 ml/min, the temperature 30 "C, and the absorbance of the effluent was recorded at 280 nm.

Ptrrificution of Glucose-1-phosphate Cytidylyltrunsferase-All proce- dures were carried out at 5 "C unless otherwise stated. The protein fraction after Sephadex G-25 gel filtration of the cell extract of a 5-liter culture (strain P9254, 11 g of cell paste) was loaded onto a 2.5 x 14-cm column of Q-Sepharose Fast Flow (Pharmacia), which had been equili- brated with buffer B. The column was washed with 300 ml of buffer B to remove unbound material. The chromatogram was then developed with a 1.0-liter linear gradient of &500 m~ NaCl in buffer B. Glucose- 1-phosphate cytidylyltransferase eluted at about 230 m NaCl in the gradient.

The fractions containing the highest amount of glucose-1-phosphate cytidylyltransferase, as judged from activity determinations, were pooled (43 ml) and dialyzed overnight against buffer C (50 m sodium phosphate buffer, pH 7.0, containing 1.7 M ammonium sulfate and 22% glycerol) The dialyzed protein fraction was divided in three aliquots, each treated separately by consecutive hydrophobic interaction chro- matography camed out at 22 "C. Each aliquot was loaded onto a column of phenyl-Superose (0.5 cm x 5 cm, HR 5/5, FPLC system, Pharmacia) which had been equilibrated with buffer C (freshly prepared). The col- umn was washed with 3 ml of buffer C to remove unbound protein. The chromatogram was then developed with a 30-ml linear decreasing gra- dient of 1.7 to 0 M ammonium sulfate in 50 m~ sodium phosphate buffer, pH 7.0, containing 22% glycerol. Glucose 1-phosphate cytidylyltransfer- ase eluted at about 0.8 M ammonium sulfate in the gradient. The frac- tions containing the highest amount of enzyme activity were pooled and passed through a Sephadex G-25 column using buffer B for equilibra- tion and elution.

The protein fraction (21 ml) after the buffer change on Sephadex G-25 was, in two consecutive runs, loaded onto a Mono Q column (0.5 x 5-cm, HR 5/5, FPLC system, Pharmacia), which had been equilibrated with buffer B. The separation was camed out at 22 "C. After sample passage the column was washed with 3 ml of buffer B, and the chro- matogram was developed with a 15-ml linear gradient of 0-0.3 M NaCl in buffer B, followed by a 5-ml linear gradient of 0.3-1.0 M NaCl in buffer B. Glucose 1-phosphate cytidylyltransferase eluted at about 240 m NaCl in the gradient. The fractions, from Mono Q chromatogra- phies, with the highest enzyme activity were pooled.

Other Procedures-The purity and M, of glucose 1-phosphate cytidy- lyltransferase under denaturating conditions were studied using poly- acrylamide gel electrophoresis in presence of sodium dodecyl sulfate (SDS-PAGE), performed according to the manufacturers (Bio-Rad) di- rections using gradient gels (4-20% acrylamide) and the Mini Ptotean I1 electrophoresis equipment.

Amino-terminal sequencing of purified glucose 1-phosphate cytidyl- yltransferase was performed on samples (approximately 30 ng) which had been passed through a Sephadex G-25 column (0.6 x 3.9 cm) using 0.1 M ammonium acetate, pH 7, as eluent. The amino-terminal protein sequence was determined with an Applied Biosystem model 470A se- quencer equipped with a 120A on-line phenylthiohydantoin analyzer.

Nucleotide sugars synthesized by alternative substrates were ana- lyzed by gas chromatography-mass spectrometry after hydrolysis and conversion of the sugar to alditol acetates as described earlier (2).

RESULTS

Assay of Glucose-1 -phosphate Cytidylyltransferase-A new HPLC assay method was developed for determination of glu- cose-1-phosphate cytidylyltransferase by CDP-D-glucose forma- tion from a-D-glucose l-phosphate and CTP. The CDP-D-glucose (retention time; 8.2 min) product is less acidic than CTP (13.2 min), because of the number of phosphates and so has a shorter retention time on the LC-SAX anion exchange HPLC column. Also the breakdown or contaminating products, CMP (5.4 min) and CDP (9.8 min), are separated from the CTP substrate and the enzyme reaction product. The time course of CDP-D-glucose formation by glucose 1-phosphate cytidylyltransferase only slightly deviates from linearity for up to 40% of the starting amount of CTP. Over the range used, there was a linear rela- tionship between initial velocity and amount of enzyme.

The same HPLC pattern was obtained when the samples were analyzed for up to 4 days after termination of the reaction. Furthermore, there was no change in concentration of CMP and CDP during the assay period, except in analysis of crude

124 Glucose-1-phosphate Cytidylyltransferase of Salmonella

TABLE I Bacterial strains and plasmids used

Straidplasmid Relevant genotypelphenotype Ref. or 8OuTce

S. enterica type B

S. enterica LT2 S1520/88 Clinical isolate"

P9029 A(hisD-medG)388 -254

W 7 4 (P4052) A(sbcB-rfb) 9

4 BO29 carrying pPR945

E. coli K12 strain

Plasmid pPR945 Derivative from pPR645 (8)

with 8.5-kilobase insert from positions 2.9 to 11.4, which carries first seven genes of the rfb gene cluster of S. enterica strain LT2, in vector pKOl

" Clinical isolates were from the National Bacteriological Laboratow, Stockholm, Sweden.

enzyme preparations (see below). For a fresh CTP solution the content of CMP was less than 0.5% and the content of CDP approximately 6%. The total absorbance area of CTP and CDP- D-glucose was constant over the range between 0% and over 90% conversion. Thus, CTP and CDP-D-glucose seem to have similar, or the same, molar absorption coefficients. With the HPLC method for assay of the glucose-1-phosphate cytidylyl- transferase activity of crude enzyme preparations, it was shown that only a small part of CTP was utilized for synthesis of CDP-D-glucose whereas most, 50-80%, of CTP was hydro- lyzed to CDP. Only strain P9254 carrying plasmid pPR945 (Table I), showed a high glucose-1-phosphate cytidylyltransfer- ase activity beside the hydrolysis of CTP.

Purification and Physical Characterization of Glucose-l- phosphate Cytidylyltransfemse-Glucose-1-phosphate cytidyl- yltransferase (rfbF) was purified from the overproducing strain (P9254) by three chromatography steps. In the first step, ion exchange chromatography, rfbF was separated from most other proteins. No formation of CMP or CDP from CTP was detected in any fraction of the chromatogram. Already afier the second step, FPLC phenyl-Superose, the rfbF was more than 90% pure (Table 11). SDS-PAGE analysis of the pooled fractions gener- ated after the last step is presented in Fig. 1. By Coomassie Blue staining &r SDS-PAGE trace amount of contaminators at the M, 59,000 and 80,000 could be detected on heavily loaded gels. The enzyme preparation was estimated to be about 95% pure, and the subunit M, for the rfbF estimated to 31,000 2 2,000. Automated Edman degradation of the purified rfbF yielded the following NH2-terminal sequence: NH,-M-K-A-V-I-

Steady State Kinetic Characterization and Substrate Speci- ficity of Glucose-1-phosphate Cytidylyltransferase-Double-re- ciprocal plots of initial velocity as a function either of CTP concentration (in the range of 0.2-3 m ~ ) at several levels of a-D-glucose 1-phosphate concentration (0.4-10 m ~ ) or of a-D- glucose 1-phosphate concentration at several levels of CTP con- centration yielded apparently parallel lines. Such patterns are indicative of a ping-pong mechanism according to the rate law

L-A-GGL-G-T-R-L-S-E-E-T-I-V-K-P-.

v = V-L4XBI/(K,,*[Bl+ K,,,[AI + [AI[BI) (Eq. 3)

While maintaining substrates concentrations at a constant ratio, the double-reciprocal plots yielded linear curves of initial velocity as a function of CTP concentration (0.23 m ~ ) for the forward reaction and as a function of CDP-D-glucose concentra- tion (0.15-2.5 m ~ ) for the reverse reaction. This is in agree- ment with the suggested ping-pong mechanism, whereas a se-

quential mechanism would not have given linearity. In both graphs the curves converged to a single llv-axis intercept, the reciprocal of V,, for the forward and the reverse reaction, respectively.

In the range of concentrations used, pyrophosphate (0.6-1.8 r n ~ ) is a noncompetitive type inhibitor with respect to CTP (0.3-1.8 m ~ ) and competitive with respect to a-D-glucose 1-phosphate (0.33-2.0 m ~ ) . With regard to CDP-D-glucose (0.33-0.67 111~) as a product inhibitor, the inhibition patterns appears to be competitive with CTP and a noncompetitive with respect to a-D-glucose 1-phosphate in the range of concentra- tions used. The values of the slope and intercept inhibition constants, Ki, and ICii are given in Table 111.

All the initial velocity data can be described using Equation 1, and the values of the kinetic parameters given in Tables I11 and IV.

In addition to the formation of CDP-D-glucose (determined as CDP-~-[~~C]glucose) also CMP increased during the reactions with CDP-D-glucose as a product inhibitor. The initial rate of CMP formation increased with increased concentration of CDP- D-glucose and decreased with increased concentration of CTP (Fig. 2a). At various concentration of a-D-ghCOSe 1-phosphate, and a constant concentration of CTP, there was no significant change in the rate of CMP formation (Fig. 2b).

The specificity of the purified enzyme for various nucleoside triphosphates and sugar 1-phosphates in the forward reaction and nucleotide sugars in the reverse reaction was studied using an assay system in which inorganic pyrophosphatase was omit- ted. By slight changes of the potassium phosphate gradient in the HPLC, the different nucleoside mono-, di-, and triphos- phates and the nucleotide sugar were resolved (Table V). In the forward reaction the highest activity was obtained with the combination of CTP and a-D-glucose 1-phosphate, and only 5.4% of that activity was obtained with the substrates UTP/a- D-glucose 1-phosphate. Exchange of a-D-glucose 1-phosphate to a-D-glucosamine 1-phosphate or a-D-xylose 1-phosphate yielded 15 and 4.6% activity, respectively, in combination with CTP, but no significant activity was detected in combination with UTP. With a-D-galactose 1-phosphate, a-D-mannose 1-phosphate, N-acetyl-a-D-glucosamine 1-phosphate, or D-ribose 1-phosphate as sugar donors, no significant activity was obtained. Neither was any activity obtained when ATP, GTP, ITP, or dTTP was used as nucleotide substrates. In the reverse reaction activity was obtained only with CDP-D-glucose as nucleotide sugar.

In absence of pyrophosphate CDP-~-[~~C]glucose was con- verted to a-~-[~~C]glucose 1-phosphate with concomitant for- mation of CMP, but no significant increase of CMP was found in a reaction with CTP as a single substrate (Table V).

The conversion of CTP and one or the other of a-o-glucosa- mine 1-phosphate or a-D-xylose 1-phosphate were followed in prolonged incubations using same conditions as described for preparation of CDP-~-['~C]glucose (see "Materials and Meth- ods"). At about 75% conversion of the sugar phosphate the reaction was stopped, and the formed CDP-sugar was purified by HPLC. After hydrolysis and conversion to alditol acetates the sugar was analyzed by gas chromatography-mass spec- trometry. From the reaction with a-D-glucosamine 1-phosphate as sugar donor the main GC-peak, 70.5%, was identified by MS as glycosamine (not shown). Correspondingly, from the reaction with a-D-xylose 1-phosphate as sugar donor the main GC-peak, 71.1%, was identified as xylose (not shown). Also the conversion of UTP and a-o-glucose 1-phosphate was studied in prolonged incubations. The area of the HPLC peak which increased dur- ing the reaction increased without any symmetric change when the samples were 'spiked" with commercially available UDP- D-glucose.

Glucose-1-phosphate Cytidylyltransferase of Salmonella 125 TABLE I1

Purification of glucose-I-phosphate cytidylyltransferase from S. enterica strain P9254

s tep Procedure

_______~

protein Tota! T o t a l Purification Yield

activity Specific activity

mg units unitslmg -fold % 1. Sephadex G-25 250 40.4 0.16 1.0 100 2. Q-Sepharose Fast Flow 27.5 34.3 1.33 8.3 84.9 3. FPLC phenyl-Superose 2.65 25.8 9.73 61 63.9 4. FPLC Mono Q 2.06 21.3 10.3 64 52.7

0.2 0.2 E .- a b

c. "

FIG. 1. SDS-PAGE showing the subunit of purified enzymes. Electrophoresis of glucose-1-phosphate cytidylyltransferase (1.3 pg, right) and Bio-Rad low molecular weight calibration kit proteins (left ), from top; phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin (44,000), carbonic anhydrase (30,000), soybean trypsin in- hibitor (21,0001, and lysozyme (14,4001, on a linear (620%) gradient acrylamide gel. Staining: Ccamassie Brilliant Blue.

TABLE I11 Inhibition constants, Kis and Kib of glucose-1-phosphate

cytidylyltransferase for CDP-D-ghcose and pyrophosphate at different concentration of CTP and a-D-glucose 1-phosphate (Glc-1-P)

ICTpl

0.30 0.60 1.78

Various

mM

0.30 0.60 1.78

Various

[Glc-1-PI

mM Various

0.33 0.67 2.00

Various

0.33 0.67 2.00

Inhibitor Ki.

mM CDP-Glc 0.067"

0.133 f 0.022 0.430 t 0.046 0.031 f 0.001 0.049 t 0.002 0.113 -c 0.017

0.543 2 0.109 0.833 ? 0.117 0.256 = 0.051

0.573" 1.983 r 0.483

PP, 0.441 f 0.124

Kii

mM 0.60"

0.73 f 0.25 1.48 f 0.19

1.64 f 0.47 2.25"

3.50 & 0.30 " Standard error not given.

TABLE TV Kinetic properties of glucose-1-phosphate cytidylyltransferase

from S. enterica strain P9254

mM CTP 0.28 ? 0.01 a-wGlucose 1-phosphate 0.64 2 0.05 11.4 f 0.2 CDP-D-glucose F'yrophosphate

0.11 2 0.04 1.89 ? 0.30 24.6 ? 2.3

pmollminlmg

DISCUSSION Although other assay methods exist for determination of glu-

cose 1-phosphate cytidylyltransferase activity (131, only analy- sis by liquid chromatography can provide (i) specificity; (ii) separation of metabolites, contaminants, and breakdown prod-

[CTPI. mM [G-I-PI. m M

FIG. 2. The initial rate of CMP formation in product inhibition by CDP-mglucose of the forward glucose-1-phosphate cytidylyl- transferase reaction. CDP-D-glucose at 0.33 xm (0) or 0.67 m w (0) was included in forward reactions with 0.003 units of glucose-l-phos- phate cytidylyltransferase at 0.67 m~ a-wglucose 1-phosphate and vari- ous concentration of CTP (a) and at 0.60 m~ CTP and various concen- tration of a-o-glucose 1-phosphate (b).

ucts; and (iii) a precise quantitation. The HPLC assay we have developed has significant advantages over previously described procedures, since it detects all the nucleotide compounds in- volved, including the breakdown products of the nucleoside triphosphate. The assay permitted detection of the rapid break- down of CTP into CDP when unpurified enzyme extracts were used; this breakdown greatly affects the quantitative analysis of the enzyme and would result in very low yields if a crude enzyme preparation were used for synthesis of CDP-D-glucose. In the present study no such breakdown was detected even after the first step of enzyme purification.

We also succeeded in purification of the Salmonella group B rfbF, which was greatly facilitated by the cloning of the gene encoding this enzyme. Without the amplification provided by cloning i t would have been difficult to purify the small amounts of enzyme found in wild-type S. enterica cells. The specific activity is about 60 times higher for the enzyme presented in this report compared with the purified glucose-1-phosphate cy- tidylyltransferase from serovarparatyphi A( 13). The cloned rfb fragment codes for and express rfbF plus several other pro- teins. Thus, in the cell-free extract of strain P9254 carrying the cloned genes, rfbF accounted for approximately 1.5% of the protein content (3.8 mg/5 liters of culture, calculated on basis of the specific activities of pure enzyme). Already after the second step of purification (chromatography on basis of hydrophobic- ity) the rfbF was more than 90% pure (Fig. 1, Table 11). As more than 50% of the total rfbF could be recovered in pure form, the final yield was approximately 2 mg.

The glucose-1-phosphate cytidylyltransferase catalyzes a re- versible bimolecular group transfer reaction. Our steady state initial rate studies, including product inhibition patterns, are consistent with a double displacement or "ping-pong Bi Bi" pathway (14) involving a cytidylylenzyme (Fig. 3). This assign- ment is supported by the formation of a-~-['~C]glucose l-phos- phate from CDP-~-['~C]glucose in the absence of pyrophos- phate. This mechanism is different from that of glucose-l- phosphate uridylyltransferase (EC 2.7.7.9) from human

126 Glucose-1-phosphate Cytidylyltransferase of Salmonella TABLE V

Substrate specificity of glucose-1-phosphate cytidylyltransferase from S. enterica strain P9254

Purified glucose-1-phosphate cytidylyltransferase (0.03 units) was in- cubated with substrate A (2.0 n") and substrate B (6.0 n") in 300 pl of 50 m~ Tris-HC1, pH 8.0, containing 12 n" MgC12.

Substrate A Substrate B velocity Initial

Forward reaction: ATP CTP CTP CTP CTP CTP CTP CTP CTP ITP GTP GTP dlTP UTFJ UTP UTP

a-u-Glucose 1-phosphate a-u-Glucose 1-phosphate a-u-Galactose 1-phosphate a-u-Mannose 1-phosphate a-u-Glucosamine 1-phosphate N-Acetyl-a-D-glucosamine 1-phosphate a-u-Xylose 1-phosphate

None u-Ribose 1-phosphate

a-u-Glucose 1-phosphate a-u-Glucose 1-phosphate a-u-Mannose 1-phosphate a-u-Glucose 1-phosphate a-D-Glucose 1-phosphate a-u-Glucosamine-1-phosphate a-u-Xylose-1-phosphate

relative

10.001 1.00

10.001 10.001

0.15 10.001

0.046 10.001 10.001" 10.001 10.001 10.001 10.001 0.054

10.001 10.001

Reverse reaction: ADP-u-glucose Pyrophosphate 10.001 CDP-u-glucose Pyrophosphate 2.1 CDP-o-glucose None 0.24b dTDP-u-glucose Pyrophosphate 10.001 dTDP-L-rhamnose Pyrophosphate 10.001 UDP-u-glucose Pyrophosphate 10.001 UDP-u-galactose Pyrophosphate 10.001 UDP-u-mannose Pyrophosphate 10.001 UDP-N-acetyl-u- F'yrophosphate 10.001

glucosamine

No sigdicant increase of CMP or CDP was detected during the

Determined both as [l*Clglucose 1-phosphate and CMP, which were incubation period.

formed concomitantly.

G-I-P

FIG. 3. The individual steps of glucose-1-phosphate cytidylyl- transferase ( E ) reaction in a ping-pong Bi Bi system. G-1-P; a-u- glucose 1-phosphate, CDPG, CDP-D-glucose, PP,, inorganic pyrophos- phate and E-CP cytidylylenzyme.

erythrocytes which has been found to be sequential (Ordered Bi Bi) (15, 16). CMP is formed in the reverse reactions if pyro- phosphate is absent (Table V) or in reactions where the con- centration of CDP-mglucose is high compared with pyrophos- phate concentration or the concentrations of CTP. No formation

of CMP was found at the initial stage of either the forward or the reverse reaction, neither in the forward reaction when py- rophosphate was added as a product inhibitor. However, with CDP-D-glucose as a product inhibitor, CMP was formed already at the initial stage of the reaction (Fig. 2). Thus we conclude that water competes with pyrophosphate and CTP, and thereby the cytidylylenzyme is hydrolyzed. The reaction can be de- scribed as follows.

Cytidylyl-E + H,O + CMP + E

REACTION 1

The purified glucose-1-phosphate cytidylyltransferase turned out to be very specific (Table V). However, the substrate specificity is somewhat different from glucose-1-phosphate cy- tidylyltransferase from serovar paratyphi A, which was re- ported to have no activity with a-D-glucosamine 1-phosphate but some activity with a-D-mannose 1-phosphate (13). The rfiF genes from representative strains of Salmonella groups A, B, and D l appear to be identical based on restriction map analysis (171, and it would be surprising if there were significant differ- ences in specificity.

The NHz-terminal sequence of the purified rfbF agrees com- pletely with the sequence predicted from the nucleotide se- quence of the Salmonella group B rfbF gene for codons 1-22 (5). Also the SDS-PAGE estimated subunit M , (31,000) agrees well with the M , of 29,035, calculated from the amino acid compo- sition deduced from the nucleotide sequence of the rfbF gene.

Acknowledgment-We acknowledge the expert assistance by Dr. M. Jerling in the statistical treatment of kinetic data.

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