ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 296, No. 1, July, pp. 122-128, 1992

Cloning, Expression, and Nucleotide Sequence of g/gC Gene from an Allosteric Mutant of Escherichia co/i 6’ Paritosh Ghosh,2 Christopher Meyer, Elaine Remy, Doug Peterson, and Jack Preiss3 Department

of Biochemistry,

Received November

Michigan

State University,

East Lansing, Michigan

48824

25, 1991, and in revised form March 2, 1992

The Eecherichia coli B mutant strain CL1136 accumulates glycogen at a 3.4- to 4-fold greater rate than the parent E. coli B strain and contains an ADPglucose synthetase with altered kinetic and allosteric properties. The enzyme from CL1136 is less dependent on the allosteric activator, fructose 1,8bisphosphate, for activity and less sensitive to inhibition by AMP than the parent strain enzyme. The structural gene, glgc, for the allosteric mutant enzyme was selected by colony hybridization and cloned into the bacterial plasmid pBR322 by insertion of the chromosomal DNA at the PsA site. One recombinant plasmid, designated pKG3, was isolated from the genomic library of CL1 136 containing g&C. The cloned ADPglucase synthetase from the mutant CL1 136 was expressed and characterized with respect to kinetic and allosteric properties and found to be identical to the enzyme purified from the CL1 136 strain. The mutant g&C! was then subcloned into pUC118/119 for dideoxy sequencing of both strands. The mutant g&C sequence was found to differ from the wild-type at the deduced amino acid residue 67 where a single point mutation resulted in a change from arginine to cysteine. 0 1992 Academic Press. Inc.

ADPglucose synthetase, the gene product of gZgC, catalyzes the formation of ADPglucose from glucose l-phosphate and ATP and is the key regulatory enzyme in the biosynthesis of bacterial glycogen. ADPglucose serves as a glucosyl donor to a glycogen or maltodextrin primer (l4) and the reaction is catalyzed by glycogen synthase, the glgA gene product. Previous studies have shown that the regulation of bacterial glycogen synthesis occurs at the level of ADPglucose synthesis (l-7). The altered rate of glycogen accumulation in mutant strains of Escherichia coli, at either a faster (8-10) or a slower rate (10) than ’ The research reported herein was supported by Grant AI 022385 from the National Institutes of Health. s Current address: National Cancer Institute-F.C.R.D.C., P.O. Box B, Frederick, MD 21702. 3 To whom correspondence should be addressed.

that in the parent strain, has been shown to be due to the altered regulatory properties of ADPglucose synthetase in those mutants. An E. coli B mutant strain, designated CL1136, accumulates glycogen at four times the rate observed for the parent E. coli B strain (9). This mutant contains normal glycogen synthase and branching enzyme activity but has an ADPglucose synthetase with altered kinetic and allosteric properties. The mutant enzyme is less dependent on the allosteric activator, fructose 1,6-bisphosphate, and has a lower apparent affinity for the inhibitor, AMP, than does the parent enzyme. To obtain information on the nature of the amino acid substitution(s) caused by the mutation and to gain new knowledge about the structure-function relationships between the regulatory sites, the CL1136 mutant ADPglucase synthetase was cloned. This report describes the cloning and expression of the mutant gene, the kinetic and allosteric properties of the cloned CL1136 enzyme, and the nucleotide sequence of the CL1136 glgC gene. EXPERIMENTAL

PROCEDURES

Reagents. The Random Primed Labeling Kit was purchased from Boehringer Mannheim Biochemicals. [32P]dCTP (3000-4000 Ci/mmol) was obtained from Amersham (Arlington Heights, IL 60005). A colony/ plaque hybridization transfer membrane was purchased from NEN Research Products. Acrylamide, iV,iV’-methylene bisacrylamide, ampicillin, tetracycline, isopropyl P-D-galactopyranoside, 5’-bromo-4-chloro-3-indolyl P-D-galactopyranoside were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents were obtained at the highest possible purity. Bacterial strains and media. Bacterial strains were E. coli K12 G6MD3 (Hfr, his, thi, Str”, A(malA-asd)) (12), HBlOl (F-, hsd20&, mi), SupE44, ara14, y-, galK2, lacY1, proA2, rpsL20, ~~1-5, mtl-1, recA13, (SL)) (13), and MV1193 (A(lac-proAB), rpsL, thi, endA, spcB15, hsdR4, A(srl-recA)306::TnlO(tet’) [B(traD36, proAB+, lacIq, lacZAM15)]) (14) and DH5a (15). LB medium contained 1.0% tryptone, 0.5% yeast extract, 1.0% N&I, and 0.2% glucose. Kornberg media contained 1.1% K,HPO,, 0.85% KH,PO,, 0.6% yeast extract, and 0.2% glucose (in solid medium 1% glucose added). Solid media were made by adding 1.5% agar. Drug selection plates contained 12.5 rg/ml of tetracycline or 100 pg/ml of ampicillin. Diaminopimelic acid was added at 50 @g/ml for cells lacking the asd gene.

122

0003-9861/92 All

$5.00

Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.

CLONING

OF MUTANT

Restriction enzymes. Restriction endonucleases, E. coli Klenow fragment, T4 DNA ligase, and calf intestine alkaline phosphatase were obtained from Bethesda Research Laboratories, Boehringer Mannheim Biochemicals, and Pharmacia PL Biochemicals. Buffers used to digest DNA were according to the manufacturer’s recommendations. Enzyme assay. For purification of the enzyme, ADPglucose synthetase was measured in the pyrophosphorolysis direction (6). The reaction mixture contained in 0.25 ml, 10 pmol of Tris-Cl buffer (pH 8.5), 100 pg of BSA,4 2 pmol of MgClx, 0.2 pmol of ADPglucose, 0.5 of Fmol “‘PP, (sp act 0.5 to 3 X lo6 cpm/rmol), 0.3 pmol of fructose 1,6-Pz, 2.5 pmol of NaF, and enzyme. In the synthesis direction (6) the reaction mixture contained in 0.2 ml, 20 Nmol of Hepes-NaOH buffer (pH 7.0), 100 pg of BSA, 0.1 rmol of [“Clglucose 1-P (0.5 to 5 X lo6 cpm/Fmol), 0.3 pmol of ATP, 0.75 rmol of MgClx, 0.2 pmol of fructose 1,6-PZ, 0.9 pg of crystalline yeast inorganic pyrophosphatase and enzyme. Gel electrophoresis. Restriction endonuclease digests were mixed with loading dye (0.25% bromophenol blue, 0.25% xylene cyan01 FF, and 15% glycerol in water) and were subjected to electrophoresis in 1% agarose gel in 1X TBE buffer (90 mM Tris-90 mM boric acid-2.5 mM Na,EDTA, pH 8.3) (16). The electrophoresis was done at 50-100 V for 2-4 h, stained with ethidium bromide (5 pg/ml), and visualized by fluorescence under shortwave uv light. Isolation of chromosomnl DNA. Chromosomal DNA from the E. coli B mutant strain CL1136 was isolated as previously described (17). Construction of the library. Chromosomal DNA (5 rg) was partially digested with PstI for 2.5 min. The reaction was stopped by heating at 65°C for 10 min. Plasmid vector (69 pg), pBR322, was also digested by PstI and subsequently dephosphorylated by calf intestine alkaline phosphatase according to the protocol described by Sambrook et al. (18). After incubation, the reaction was stopped by heating the same at 68°C for 10 min. The dephosphorylated vector was then extracted as follows: phenol (TE buffer saturated; 10 mM Tris-HCl and 1 mM EDTA, pH 8,O):chloroform:isoamyl alcohol (25:24:1) extraction (two times), chloroform:isoamyl alcohol (24:l) extraction (two times), ether (water-saturated) extraction (two times). DNA was then precipitated at -20°C overnight, by adding 3 M sodium acetate (final concentration 0.3 M), 2.5 vol. of cold 95% ethanol. Centrifugation was done for 30 min in a microcentrifuge. The pellet was washed with 70% ethanol and was dried under speed vacuum. The dried sample was dissolved in sterile TE buffer. One microgram of PstI-digested chromosomal DNA was added to 1 pg of dephosphorylated P&I-digested PBR322, and the ligation reaction was done according to the procedure described by King et al. (19). After the ligation, the reaction mixture was diluted fivefold with sterile TE buffer before transformation. CaCl,-treated competent E. coli HBlOl, prepared by the modified method of Cohen et al. (20), were transformed as described by Bolivar et al. (21). Transformed cells were added to 1 ml of LB media, and were incubated for 1 h at 37°C with shaking. After incubation, cells were collected by centrifugation using a microcentrifuge and the pellet was resuspended with half of the supernatant. The cells were plated on LB media containing 12.5 pg/ml of tetracycline. After overnight growth at 37”C, transformants were grown on LB media and plasmids were isolated using the alkali lysis method (22) to determine the percentage of transformants containing the insert. Preparation of the competent cells. Competent cells of E. coli HBlOl were made by CaCl, treatment according to Cohen et al. (20) with the following modification: after harvesting, cells were resuspended with f vol of cold 100 mM MgClz and incubated on ice for 5 min. Cells were collected by centrifuging for 6 min at 5000 rpm and resuspending with f vol of cold 100 mM CaClz and was incubated on ice for 20 min. After incubation, centrifugation was done at 5000 rpm for 5 min, and cells were resuspended gently in 3 vol of cold 100 mM CaClx. For storage, 15% glycerol was added to the cell suspension and was kept at -70°C.

* Abbreviations used: BSA, bovine serum albumin; SSC, standard saline citrate; DTT, dithiothreitol; IPTG, isopropyl P-D-thiogalactoside; PEG, polyethylene glycol; FBP, fructose 1,6-bisphosphate.

Escherichia

coli glgC GENES

123

Construction of DNA hybridization probe. The DNA hybridization probe containing the wild type glgC gene was constructed by the random priming method of Feinberg and Vogelstein (23). Plasmid pOP12 containing the E. coli K12 glgA, -B, and, -C genes (17), was digested with the restriction enzyme HincII. The digested material was subjected to electrophoresis using 0.8% low melting point agarose (Sigma products) and the 1.9.kb HincII fragment was collected by excising of the gel and the gel piece was transferred into a preweighed 1.5-ml microfuge tube. Water was added at a ratio of 3 ml water/g of gel. The tube was placed in a boiling water bath for 7 min and then transferred to a 37°C water bath until the labeling reaction was initiated. The labeling reaction was according to manufacturer’s protocol, which contained 34 ~1 of melted gel, 2 ~1 reaction mixture, 5 ~1 [o-32P]dCTP (50 &i; 3000 r/mmol) and 1~1 Klenow enzyme in a total volume of 50 gl. The incubation was done at 37°C for 3 h. The reaction was stopped by heating to 68°C for 10 min. Screening of genomic library by colony hybridization. The genomic library was screened by colony hybridization according to the procedure described by the manufacturer of NEN Colony/Plaque Screen (24) with modification. Each loo-mm plate contained 500-600 colonies. After plating out the cells, the plates were incubated up to a point where colonies were starting to appear. The plates were stored at 4°C prior to lifting. Transfer of colonies to the Colony/Plaque Screen membrane was done as described in the manufacturer’s protocol. For every plate, duplicate plating was performed to identify the nonspecific signals. Each membrane with the colony side up was placed onto a LB-tetracycline plate and the plate was incubated overnight at 37°C. The membranes were soaked from the bottom in 0.75 ml of 0.5 N NaOH for 2 min (two times) and then neutralized with 0.75 ml of 1.0 M Tris-HCl, pH 7.5 for 2 min (two times). The membranes were dried at room temperature. Prehybridization was carried out at 65°C in a solution containing 1% SDS and 1 M sodium chloride. The membranes were prehybridized for 6 h with three changes of prehybridization buffer. The prehybridized membranes were subjected to hybridization in the same prehybridization buffer containing 100 pg/ml denatured salmon sperm DNA and denatured radioactive probe (lo6 cpm). The hybridization was then allowed to proceed at 65’C overnight. After hybridization, the membranes were washed twice (5 min each) in 100 ml of 2X SSC (0.3 M sodium chloride0.03 M sodium citrate), at room temperature, twice (30 min each) in 200 ml of 2X SSC containing 1% SDS at 65°C and twice (30 min each) in 100 ml of 0.1% SSC (0.015 M sodium chloride-O.0015 M sodium citrate) at room temperature with constant agitation. The membranes were dried at room temperature and exposed to Kodak XAR-5 film at -70°C. Preparation of the crude homogenates of E. coli K12 G6MD3 transformed with the cloned gene. The cell paste (0.5 g) of E. coli B G6MD3 transformed with the recombinant plasmid, pKG3 obtained from the overnight growth was suspended in 10 ml of 0.05 M glycylglycine buffer (pH 7.0) containing 5 mM dithiothreitol and 1 mM EDTA. The suspension was exposed to sonication to disrupt the cells. The sonicated sample was centrifuged at 30,OOOgfor 10 min and the supernatant was used for enzyme assay. Partial purification of the ADPglucose synthetase isolated from E. coli K12 G6MD3 transformed with pKG3. The purification procedure was similar to that previously described (10). The enzyme was stored at a concentration of 4.5 mg/ml at -70°C in 0.1 M Hepes (pH 7.5), 0.5 mM DTT, 1 mM EDTA, and 20% (w/v) glycerol. Subcloning and sequencing of the mutant ADP glucose synthetase gene. The recombinant plasmid pKG3 was digested with HincII and the 1.9-kb fragment corresponding to the glgC gene (17, 25) was cleanly excised from a 0.8% low melting point agarose gel and isolated using a NACS ion-exchange PREPAC column (BRL). The mutant glgC gene was then blunt-end ligated to dephosphorylated Hin.cII-digested pUC119 and subsequently transformed into MV1193 cells and plated on LB/ AMP in the presence of X-gal and IPTG for blue-white screening of colonies (26). The orientation of the glgC gene was determined by HindI digestion utilizing the unique Hind111 site in g&C and the polycloning site in pUC119.

124

GHOSH

- 5.0 - 4.0

- 3.0

-

of single-stranded reactions. The entire coding region of both strands was sequenced using oligonucleotide primers derived from the E. coli K12 g&C sequence (25). The 5’ end of the g&C gene on the expression plasmid pKG3 (purified by cesium chloride density gradient centrifugation) was also sequenced by the double-stranded method to verify the changes found in pUC. The use of double-stranded templates eliminated the need to generate single-stranded DNA, which saved considerable time. In our system, plasmid templates from the host strain DH5a gave less background in the sequencing reaction lanes than those from MV1193, yielding longer readable sequences. The DNA sequencing was done by the dideoxy sequencing method (28).

2.0 1.6

- 1.0

-

ET AL.

0.5

ABCDEFG FIG. 1.

Gel electrophoresis of pKG3 and pOP12 DNA digested with the endonucleases PstI, Hi&III and HincII. Plasmid DNA was isolated as described under Experimental Procedures and digested with PstI or Hind111 or Z-ZincII. The digested DNA was resolved by electrophoresis on 1% agarose gel. Lanes B, C, and F, pOP12; Lanes A, D, and G, pKG3; Lanes A and B, DNA digested with P&I; Lanes C and D, DNA digested with HingdIII; Lanes F and G, DNA digested with HincII; Lane E, 1 kb ladder. Numbers on the right indicate kilobase size.

Screening of over 20 positive colonies resulted in the successful isolation of only the reverse (with respect to the Lac promoter on pUC119) orientation of the 1.9-kb fragment, yielding two fragments of 4.5 and 0.6 kb (17, 25). Approximately 25% of the positive colonies yielded an apparently truncated proper orientation in pUC119 which by restriction analysis was missing ca. 0.5-0.6 kb from the 5’ end of the glgC gene. More rarely, an apparent forward orientation was isolated with random deletions giving an unrecognizable restriction map. Problems with maintaining certain intact g&C genes in both orientations on pUC plasmids have been encountered before (unpublished results). The forward orientation would be expected to yield a properly expressed active protein, which is apparently not favored. As ADPglucose synthetase is a key ATP-utilizing regulatory enzyme in carbon metabolism, perhaps overexpression by a high copy number plasmid such as pUC119 (as opposed to the pBR322-derived expression vector pKG3) leads to a lethal energy imbalance in the cell. In normal E. coli metabolism, the gig genes are highly regulated, being expressed in late log/ early stationary phase under a variety of conditions (2, 17). To circumvent this problem, the proper orientation was subcloned in two parts. The isolated complete reverse orientation in pUC119 was digested with HincII and HindI and the resulting 3.2-, 1.3-, and 0.6kb fragments were isolated as previously described. The 1.3-kb fragment (corresponding to the 5’ end of g.!gC) was ligated into HincII-HindIIIdigested pUC118 while the 0.6-kb fragment was religated to the digested pUC119. Positive transformants in MV1193 or DH5a! were found to contain the 4.5- and 3.8-kb recombinant plasmids corresponding to the 5’ and 3’ ends of the g&C gene in pUC118 and pUC119, respectively. Sequencing was performed with the dideoxy method using Sequenase (USB). Single-stranded DNA was prepared using the helper phage M13K07 as described (26). Double-stranded sequencing was also performedusing PEG-purifiedplasmids as template (27). Briefly, the doublestranded template was denatured by the addition of NaOH and heated at 68°C for 10 min prior to neutralization with HCl. Subsequent steps in the sequencing reactions were essentially identical to the handling

RESULTS Cloning of ADPglucose synthetase gene from E. coli B mutant strains CL1 136. Initially attempts were made to clone the g&C gene from an E. coli mutant strain by transforming E. coli K12 strain G6MD3, which contains a deletion between m&A and asd and is therefore lacking the glg genes. Since the usd gene is a neighboring gene, the gig genes could be selected by selecting for the asd

gene and then scoring for the Gig+ phenotype among Asd+ transformants, no longer requiring diaminopimelic acid for growth. However, attempts were not successful, which was in contrast to previous results (17), where glg genes were successfully cloned into pBR322. Strain HBlOl was thus selected because of its high transformation efficiency, and lack of host specific restriction-modification properties, because there was a concern of cloning E. coli B genes into an E. coli K12 strain. The cloning strategy was changed to colony hybridization. Although HBlOl has its own ADPglucose synthetase gene, which in turn gave a positive signal in colony hybridization, the intensity of the positive signal corresponding to the multicopy plasmid containing the gig gene was much greater than the signal corresponding to the cell by itself (data not shown). One colony showed the positive hybridization signal. The re-

pop12

PSt 1 FIG. 2.

blonlc(-

pKG3

Restriction endonuclease cleavage map of pOP12 and pKG3. The plasmids were digested with three restriction endonucleases which have one or two cut sites in pBR322. The map was constructed based on the size of the fragments obtained from the digest and comparison with the pOP12 restriction cleavage map previously deduced (17). The heavily shaded region is pBR322 DNA.

CLONING TABLE

OF MUTANT

Escherichia

were in agreement with the proposed orientation of the clones.

I

Expression of ADPglucosePyrophosphorylasein the E. coli K-12 Deletion Strain G6MD3 Strain

Expression of the cloned gene in E. coli K12 deletion strain G6MD3. In order to determine whether the cloned

ADPglucose pyrophosphorylase activity (am01 ATP formed/min/mg protein)

Fold activation

0.04

34”

E. coli B E. coli G6MD3 G6MD3/pKG3

1.5”

0.001 0.45

125

coli glgC GENES

’ Assay was done in the pyrophosphorolysis direction. Assay conditions were as described under Experimental Procedures.

combinant plasmid from the positive clone, designated pKG3, was isolated and retransformed into the E. coli deletion mutant G6MD3 to confirm the cotransformation with the neighboring a.sdgene. The dependency of G6MD3 on diaminopimelic acid for its growth due to the lack of usd gene was abolished upon transformation with the recombinant plasmid. Restriction enzyme mapping. It was of interest whether plasmid pKG3 was similar to pOP12, the plasmid containing all of the glycogen structural genes (16). The recombinant plasmids were analyzed with three restriction enzymes: P&I, HindIII, and HincII, and were compared to pOP12 (Fig. 1). In the PstI digests, an insert of 8.5 kb was obtained in pKG3, which was also present in pOP12. Two other fragments (0.9 and 1.6 kb) were present in pOP12, but were missing in pKG3. Okita et al. (17) have shown by deletion mapping and subcloning that the glgC gene coding for ADPglucose synthetase resides near the 8.5kb region on the pOP12 restriction map. The orientation of the inserted DNA fragment was determined by digestion with HindIII. According to the fragment size calculation based on the restriction endonuclease cleavage map of pOP12, the orientation of the insert was reverse for pKG3 with respect to pBR322 (Fig. 2). Upon digestion with H&II, pOP12 generates a fragment of 1.9 kb in size, in addition to other fragments, which contains the full length glgC gene along with the flanking regions. This 1.9-kb fragment was present in the recombinant plasmid pKG3. The other fragment sizes

gene expresses the ADPglucose synthetase, the crude homogenate of the pKG3-transformed E. coli K12 G6MD3 cell was assayed for the enzyme. Table I represents the expression of the ADPglucose synthetase in nontransformed and transformed E. coli K12 G6MD3. The high level of enzyme activity in the case of G6MD3 (pKG3), over 11-fold over the wild-type E. coli B strain, was probably due to the high copy number of the plasmid pBR322. The fold of activation by fructose 1,6-Pr for the pKG3 enzyme was found to be 1.5, which is a characteristic of the CL1136 enzyme being less dependent on the activator than the wild-type enzyme (9, 10). The fold activation for the E. coli enzyme is 30-40 (29). Characteristics of the ADP glucose synthetase isolated from extracts of E. coli K12 G6MD3 transformed with pKG3. To verify that the expressed gene was the same

as the ADPglucose synthetase previously isolated from the mutant strain CL1136, the enzyme was partially purified from extracts of transformed E. coli K12 G6MD3 as summarized in Table II. The enzyme was purified to a specific activity of 5 I.U./mg. The ca. lo-fold purification achieved is in good agreement with earlier purifications of the CL1136 enzyme taken through DEAE chromatography (9) and was suitable for detailed kinetic analysis in the synthesis direction. The partially purified pKG3 enzyme was heat sensitive and lost activity when stored in the absence of 20% (w/v) glycerol, which was identical to what had been found with the enzyme purified from CL1136 (9,10). This is in sharp contrast to the wild-type enzyme where the purification procedure includes a 65°C heat step (6). Activation by fructose 1,6-bisphosphate. The fructose 1,6-bisphosphate activation curve for the enzyme from pKG3-transformed strain G6MD3 is shown in Fig. 3. The activation curve is hyperbolic with a Hill slope of 0.94 f 0.07, and 50% maximal activation occurred at 4.7 PM f 1.9. The enzyme was quite active in the absence of the activator (Fig. 3), and only a 1.5-fold activation occurred with fructose 1,6-bisphosphate. This is in good agreement with the reported values for the CL1136 enzyme of 5.3 PM and the Hill slope (n) of 0.93 (9). The activator fructose

TABLE

II

Purification of ADPglucoseSynthetasefrom E. coli K12 G6MD3 Transformed with pKG3 Vol. Fraction

(ml)

Total protein be)

Homogenate 50-60% (NH,),SO, DEAE-Sepharose

23 10 6.5

265.5 107 29.5

Total activity (units) 141 107.3 149.2

Specific activity

(U/meprot) 0.53 1.5 5

Purification (fold) 1 2.6 9.5

Recovery (%) 100 76 106

126

GHOSH

.-.-

I

1 A.

ET AL. TABLE

Kinetic

Constants

IV

for ADPglucose

Synthetase Expressed in with pKG3

E. coli K12 G6MD3 Transformed

pKG3 ADPglucose Synthetase Substrate ATP Glucose 1-P 200.0

150.0

100.0

50.0

0.0

Fructose 1.6-bisphosphote.

WA

/.d

I

I 6.

Fru - P2 FIG. 3. A. Fructose 1,6-bisphosphate activation curve for pKG3 ADPglucose synthetase. The ADPglucose synthesis reaction mixture and assay are described under Experimental Procedures; Au is the velocity increase due to the presence of fructose-1,6-bisphosphate. B. Hill plot of the data.

1,6-bisphosphate stimulates ADPglucose synthesis catalyzed by the CL1136 enzyme 1.5- to 2-fold. The l0.s (concentration of inhibInhibition by AMP. itor giving 50% inhibition) for AMP for the KG3 enzyme in the absence of activator was found to be 8.8 f 0.9 PM (Table III), similar to that which has been reported for

TABLE

Activator None Fructose-Pp None Fructose-Pz None Fructose-Px

a The values in parentheses two experiments.

n

&U (mM) 0.41 0.14 0.023 0.023 2.1 1.8 are the standard

(O.Ol)O (0.01) (0.0) (0.0) (0.14) (0.0) deviations

2.6 1.7 1.1 1.0 5.5 5.9

(0.5) (0.4) (0.2) (0.1) (0.2) (0.0)

of at least

the CL1136 enzyme (9, 10). The activator concentration clearly modulates the sensitivity of the enzyme to AMP (Table III, Fig. 4); as the FBP concentration increases, the amount of AMP required to elicit 50% inhibition also increases, reaching nearly 600 PM in the presence of 1.5 mM FBP (Table III). These data are also in agreement with the reported values on AMP inhibition for the CL1136 enzyme (9,lO). Kinetic constants for the substrates. Table IV summarizes the kinetic constants of pKG3 ADPglucose synthetase for substrates and magnesium. In the absence of fructose 1,6-Pz, the saturation curve for ATP is sigmoidal and the concentration required for 50% of maximum velocity (S,,) is 0.41 + 0.01 mM. In the presence of 1 mM activator, the S0.5value decreased to 0.14 + 0.011 mM. A Hill plot of the data showed that the Hill constant de-

““1

III

and Inhibition Kinetic Constants pKG3 ADPglucose Synthetase Effecters

Activation

Effector

&.s (PM)

Fructose 1,6-bisphosphate Adenosine 5’-monophosphate No FBP +O.l mM FBP +0.3 mM FBP +0.5 mM FBP +1.5 mM FBP

4.7

a The assay was done in the ADPglucose scribed under Experimental Procedures.

for

b.6 (PM)

Hill ri 0.94

8.8 90 250 400 575 synthesis

direction

0.98 1.5 1.7 2.0 2.3 as de-

AMP, mM FIG. 4. Inhibition of pKG3 ADPglucose synthetase in the presence of various concentrations of fructose 1,6-bisphosphate. The enzyme was assayed in the direction of ADPglucose synthesis at pH 7.0 (Experimental Procedure). (0) curve obtained in the presence of 0.1 mM fructose-P,; (A) curve obtained in the presence of 0.3 mM fructose-Px; (Cl) curve obtained in the presence of 0.5 mM fructose-Pz. Kinetic constants obtained from this figure are shown in Table III.

CLONING

OF MUTANT

Escherichia

coli glgC GENES

127

FIG. 5. Nucleotide and deduced amino acid sequence of pKG3 g&C gene. The sequence shown is that of the antisense strand. The dots on the bases show the change in nucleotide compared to wild-type nucleotide sequence with no change in deduced amino acid, whereas the cross indicates a change in the nucleotide sequence resulting in a change in the deduced amino acid sequence.

creases from 2.8 f. 0.5 in the absence of fructose 1,6-P2 to 1.7 -t 0.4 in the presence of activator. This is in agreement with the data reported for CL1136 ADPglucose synthetase (9, 10). The MgC& and glucose 1-P curves, respectively, of pKG3 ADPglucose synthetase in the presence and absence of fructose 1,6-Pz were similar to those of the CL1136 enzyme (9, 10). Fructose 1,6-PZ had little effect on the So,5values of MgC& and glucose 1-P. The S0.5values of MgClz are 1.8 mM with a Hill constant of 5.9 f 0.07 in the presence of activator and 2.1 mM + 0.14 in the absence of activator with a Hill constant of 5.5 f 0.2. The S& value of glucose 1-P is 0.023 mM in the presence and absence of fructose 1,6-bisphosphate. Sequencing strategy. The source of DNA for sequencing was the mutant glgC gene from pKG3. As described under Materials and Methods, only the reverse orientation (with respect to the Lac promoter) was successfully subcloned into pUCll9 for single-stranded DNA production for dideoxy sequencing. The forward orientation could only be subcloned into two parts into pUC118/119. To ensure the fidelity of the subcloning steps, the mutation found in sequencing of the pUC plasmids was confirmed by double-stranded sequencing of the pKG3 expression plasmid. DNA sequence. Figure 5 shows the antisense strand nucleotide and deduced amino acid sequence of the pKG3 glgC gene. Dots appearing over bases indicate differences from the K12 strain wild-type sequence which do not result in a codon change. One codon was found to be altered in comparison to the wild-type gene. At amino acid position 67, arginine was converted to cysteine by a CGT to TGT change as seen on the antisense strand.

DISCUSSION

The ADPglucose synthetase gene (gZgC) from the allosteric mutant strain of E. coli B, designated CL1136, was isolated from the genomic library by colony hybridization. One recombinant plasmid, pKG3, was obtained that contained full length glgC gene as indicated by the restriction mapping (Fig. 1) and the preliminary expression studies (Table I). Further confirmation of the positive clone was made by transforming the pKG3 plasmid into the E. coli K12 deletion strain G6MD3. In addition with transformation, the dependency of G6MD3 on diaminopimelic acid for growth was abolished, which indicated the cotransformation of the neighboring asd gene (8). Preliminary expression data (Table I) shows the expression of glgC gene in G6MD3 transformed with pKG3. Kinetic analysis of the cloned glgC gene product was shown to have properties similar to those observed for the CL1136 mutant ADPglucose synthetase. The pKG3 ADPglucose synthetase is less dependent on the activator, fructose 1,6-bisphosphate, as the enzyme is highly active in the absence of activator (Fig. 3), characteristic of the CL1136 mutant enzyme (9, 10). Another property that distinguishes the CL1136 enzyme from the wild-type enzyme is the sensitivity of the enzyme toward AMP inhibition in the presence and absence of fructose 1,6-bisphosphate. AMP inhibition studies of pKG3 ADPglucose synthetase show that the activator protects the pKG3 ADPglucose synthetase against AMP inhibition, and the degree of protection is dependent on fructose 1,6-Pp concentration (Fig. 4 and Table III). This is also true for the CL1136 enzyme. In contrast, the wild-type enzyme requires a minimal concentration of fructose 1,6P2 (-3 PM) to make it most maximally sensitive toward

128

GHOSH

AMP inhibition (9, 29). However higher concentrations of fructose 1,6-Pz (>3 PM) cause the wild-type enzyme to be also less sensitive to AMP inhibition (6, 7). Sequencing of the subcloned glgC gene revealed one amino acid change, arginine 67 to cysteine, when compared to the wild-type. This is consistent with earlier physical data on the CL1136 enzyme: the N-terminal and first 48 amino acids, C-terminal, and molecular weight were the same as the wild-type enzyme (10). To ensure that the mutation was not an artifact of subcloning, the codon change was confirmed by double-stranded sequencing of the expression plasmid pKG3 (see Materials and Methods, Results sections). In terms of structure/function relationships, it may be significant that the mutation is in the N-terminal end of the protein as this area has been shown to be important in activator binding (30, 31). Such a change disrupts a positively charged Arg-Arg stretch, most likely to be at or near the surface of the protein, which could be involved in phosphate binding site(s) (32) for the charged effector molecules. Early chemical modification studies indicated that an arginine residue played a role in the binding of fructose 1,6-Pz (33). Perhaps the change to a neutral amino acid results in a conformational change which mimics the effect of fructose 1,6-PZbinding. Such a change could also disrupt AMP binding as this region was found to be one site of 8-azido-[3H]AMP incorporation which could be inhibited by AMP (34). Such a dual effect of the mutation on the allosteric properties of the enzyme is consistent with the idea of partially overlapping binding sites for activator and inhibitor (34). The observed reduced stability of the enzyme might be expected with the introduction of a cysteine residue near the surface of the protein where it might be subject to oxidation or unfavorable disulfide bond formation. It would be of interest to determine the effects of other amino acids at position 67 with respect to the regulatory properties of the E. coli ADPglucose synthetase. In this respect, it would also be of interest to know the location of the amino acid changes in other mutant ADPglucose synthetases having altered regulatory properties (7, 11, 35).

Top. Cell. Regul. 1, 125-160.

2. Preiss, J., and Romeo, T. (1989) in Advances in Microbial Physiology, (Rose, A., and Tempest, D., Eds.), Vol. 30, pp. 183-238, Academic Press, San Diego. 3. Preiss, J. (1978) Adv. Enzymol. Relut. Areas Biol. 46, 317-381. 4. Preiss, J. (1973) in The Enzymes (Boyer, P. D., Ed.), 3rd ed., Vol. 8, pp. 73-119, Academic Press, New York. 5. Shen, L., and Preiss, J. (1964) Biochem. Biophys. Res. Commun.

17,424-429.

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E., and Gentner,

N. (1966) Bio-

chemistry 6,1833-1845. 7. Govons, S., Gentner, N., Greenberg, E., and Preiss, J. (1973) J. Biol. Chem. 248,1731-1740. 8. Creuzat-Signal, N., Latil-Damotte, M., Cattaneo, J., and Puig, J. (1972) Biochemistry of the Glycoside Linkage (Piras, R., and Points, M. G., Eds.), pp. 647-680, Academic Press, New York. 9. Preiss, J., Lammel, C., and Greenberg, E. (1976) Arch. Biochem. Biophys. 174,105-119. 10. Kappel, W. K., and Preiss, J. (1981) Arch. Biochem. Biophys. 209, 15-28. 11. Preiss, J., Greenberg, E., and Sabraw, A. (1975) J. Biol. Chem. 260, 7631-7638. 12. Schwartz, M. (1966) J. Bacterial. 92, 1083-1089. 13. Boyer, H. W., and Roulland-Dussoix, D. (1969) J. Mol. Biol. 41,

459. 14. Zoller, M. J., and Smith, M. (1987) in Methods in Enzymology (Wu, R., and Grossman, L., Eds.), Vol. 154, p. 329, Academic Press, San Diego. 15. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) J. Biol. Chem. 3, AlO. 16. Bolivar, F., Rodriguez, R., Betlach, M., and Boyer, H. (1977) Gene 2, 75-93. 17. Okita, T. W., Rodriguez, L. R., and Preiss, J. (1981) J. Biol. Chem. 256,6944-6952. 18. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1, pp. 1.60-1.61. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 19. King, V. P., and Blakesley, W. R. (1986) Focw 8(l), l-3. 20. Cohen, S., Chang, A., Royer, H., and Heling, R. (1973) Proc. Natl.

Acad. Sci. USA 70,3240-3244. 21. Bolivar, F., Rodriguez, R., Green, P., Betlach, M., Heyneker, H., Crosa, J. H., and Falkow, S. (1977) Gene 2,95-113. 22. Clewell, D. (1972) J. Bacterial. 110, 667-676. 23. Feinberg, P. A., and Vogelstein, B. (1983) Anal. Bhchem. 132,613. 24. Colony/Plaque Screen Hybridization Transfer Membrane, Biotechnology Systems, NEN Research Products (Catalog No. NEF978/978A). 25. Baecker, P. A., Furlong, C. E., and Preiss, J. (1983) J. Biol. Chem.

258,5084-5088. 26. Vieira, J., and Messing, J. (1987) in Methods in Enzymology

(Wu, R., and Grossman, L., Eds.), Vol. 153, pp. 3-11, Academic Press, San Diego. 27. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1, pp. 1.40-1.41, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 28. Sanger, F., Milkle, S., and Coulson, A. R. (1977) Proc. Natl. Acad.

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196,884~886. 33. Carlson, C. A., and Preiss, J. (1982) Biochemistry 21,1929-1934. 34. Larsen, C. E., Lee, Y. M., and Preiss, J. (1986) J. Biol. Chem. 261, 15,402-15,409. 35. Steiner, E. K., and Preisa, J. (1977) J. Biol. Chem. 129, 246-253.