Biochimica et Biophysica Acta, 453 (1976) 418-425

© Elsevier/North-Holland Biomedical Press BBA 37506 UDP-GLUCOSE D E H Y D R O G E N A S E FROM E S C H E R I C H I A COL1 P U R I F I C A T I O N A N D SUBUNIT S T R U C T U R E

JULIAN G. SCHILLER*, FRANGOIS LAMY**, RODERICK FRAZIER*** and DAVID S. FEINGOLD Department of Microbiology, School of Medicine, Unirersity of Pittsburgh, Pittsburgh, Pa. 15261 (U.S.A.)

(Received Apri 9th, 1976)

SUMMARY UDPglucose dehydrogenase from Escherichia coli has been purified 330-fold with an overall yield of 27 ~. A single homogeneous subunit was demonstrated by ultracentrifugation in 6 M guanidium chloride and by dodecyl sulfate-polyacrylamide gel electrophoresis. Since the molecular weight of the intact dehydrogenase is in the order of 86 000 and the subunit weight determined by the dodecyl sulfate-polyacrylamide gel electrophoresis is 47 000, the enzyme consists of two polypeptide chains. The sole amino terminal acid shown by the dansylation technique was arginine. Forty-four tryptic peptides were obtained by peptide mapping, in agreement with the number of arginine and lysine residues/mole protein [43] determined by amino acid analysis. The data are consistent with the presence of two identical or very similar polypeptide chains in E. coli UDPglucose dehydrogenase.

INTRODUCTION UDPglucose dehydrogenase (UDPGIc :NAD oxidoreductase, EC 1.1.1.22) has been purified to homogeneity from extracts of bovine liver and shown to consist of 6 identical subunits of molecular weight 52 000 [1, 2]. The dehydrogenase also has been purified partially from a number of eukaryotic and prokaryotic organisms, among others a derepressed strain of Escherichia coli [3]. The latter enzyme differs from the liver UDPglucose dehydrogenase in regard to its kinetic properties and also seems to have a much lower molecular weight than the mammalian enzyme. We now have obtained the E. coli UDPglucose dehydrogenase as a homogeneous protein and

Present address: Department of Chemical Engineering, University of Pittsburgh, Pittsburgh, Pa. 15261, U.S.A. "* Ddpartement de Biochimie, Universitd de Sherbrooke, Sherbrooke, Qudbec, Canada. "*" Present address: School of Dental Medicine, Howard University, Washington, D.C. 20001, U.S.A. "

419 have shown that it probably consists of two identical subunits. These results are presented in this paper. MATERIALS AND METHODS

Materials UDPglucose and NAD were purchased from the Sigma Chemical Co., St. Louis, Mo. Pure enzymes for use as molecular weight markers were obtained from Worthington Biochemical Corp., Freehold, N.J. All other materials used were described previously [3].

Methods Enzyme assay. UDPglucose dehydrogenase was assayed as described previously [3]. A unit of enzyme activity is defined as the amount of enzyme required to produce 2 #mol of N A D H (equivalent to the oxidation of 1 /~mol of UDPglucose) per min at 30 °C. Disc gel electrophoresis. Polyacrylamide disc gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol was done as described by Weber and Osborn [4], using bovine serum albumin, chymotrypsinogen A, and egg white lysozyme as molecular weight standards. 50-~1 samples containing 5 to 20 ~g protein were layered on 10~o gels (3 cm long, 0.6 cm diameter) and subjected to electrophoresis at 8 mA per gel for 5 h or until the marker dye had migrated to the end of the tube. The gels were fixed, stained, and scanned with a Gilford spectrophotometer at 550 nm. Absorption spectrum. The absorption spectrum of the UDPglucose dehydrogenase was recorded in a Cary Model 14 spectrophotometer in a cuvette with a 1 cm light path. The enzyme (1.15 mg/ml) [5] was dissolved in 50 mM potassium phosphate, pH 7.0, 1 mM in dithiothreitol. The same buffer was used as a blank. Ultracentrifugation. Sedimentation velocity studies were carried out in a Spinco Model E ultracentrifuge at 20 °C and a speed of 52 000 rev./min. The enzyme was present in a concentration of 3 mg/ml in buffer. UDP-glucose dehydrogenase also was examined by ultracentrifugation in 6 M guanidinium hydrochloride by the meniscus depletion method of Yphantis [6] at 26 000 rev./min and 16.6 °C. The partial specific volume calculated from the amino acid composition was corrected for the effect of 6.0 M guanidinium chloride according to Hade and Tanford [7]. Peptide "mapping". A solution of UDPglucose dehydrogenase was dialyzed against distilled water and lyophilized preparatory to reduction and S-carboxymethylation according to the procedure of Crestfield et al. [8]. The urea solution of reduced and carboxymethylated protein was dialyzed for 3 days in the dark at 4 °C against 400 volumes of distilled water each day, and the retentate was lyophilized. A portion of this material (1.2 mg) was mixed with 0.25 ml of 0.1 M (NH4)2COa containing 12/~g of TPCK-trypsin, the mixture was maintained at 22-25 °C for 16 h and then dried in a stream of nitrogen gas. The dried peptide digest was redissolved in 40 #1 of water and 10 #1 of the solution was applied to the corner of a 20 × 25 cm sheet of Brinkman polygram Gel G (Brinkman Instruments, Inc., Westbury, N.Y.). Electrophoresis was done in the long dimension of the plate at 2 kV (40 mA) for 70 min in pyridine/acetic acid/water (1:10:89, v/v/v) [9] on a Savant flat plate electro-

420 phoresis unit (Savant Instruments, Hicksville, N.Y.). The electropherogram was airdried and subjected to chromatography in the perpendicular dimension in butanol/ acetic acid/water (12:3:5, v/v/v) [9]. The gel plate was air-dried and the peptides were revealed by spraying with a 1 ~o solution of ninhydrin in 1-butanol. Protein concentration. Protein concentrations were determined as described by Lowry et al. [5]. Amino acid analysis. The enzyme solution was dialyzed exhaustively against 104 volumes of distilled water and the retentate was lyophilized. The dried enzyme (2.5 rag) was suspended in 5 ml of 6 N HCI and hydrolyzed under vacuum at 105 °C. Duplicate hydrolyses were done for 24, 36, 68, and 96 h. The HC1 was removed under vacuum and the residue was dissolved in 2.5 ml of 0.2 M citrate buffer, pH 2.2, preparatory to amino acid analysis in a Spinco Instruments Co. (Palo Alto, Calif.) model 120C amino acid analyzer. The moles of amino acid/tool enzyme ratio was calculated by assuming a molecular weight of 94 000. The tyrosine content was confirmed by estimation in alkaline solution and the tryptophan content was estimated spectrophotometrically according to Edelhoch [10]. Analysis for N terminal group. Lyophilized enzyme was treated with dansyl chloride by the method of Gray [11 ] modified as follows. Enzyme (150/~g) was taken up in 100 ,ul of 1 ~ sodium dodecyl sulfate; 100/~l of N-ethylmorpholine and 150 ¢tl of 1 ~ dansyl chloride was added. After 18 h the dansylated protein was precipitated with acetone and the insoluble residue was extracted with 1 0 ~ acetic acid. This procedure removed most of the soluble reaction products without solubilizing the protein. The dansylated protein was hydrolyzed for 4 h at 105 °C in 6 N HC1 and the hydrolyzate was examined chromatographically in two dimensions on 5 × 5 polyamide sheets as described by Woods and Wang [12]. A standard mixture of dansylated amino acids was run for comparison. Revised cell growth and enzyme purification scheme. Cells of E. coli strain MC 153 were grown as previously described [3]. Cell growth was started from a lyophilized culture each time a batch of enzyme was prepared. The purification was done as described previously except that all buffers were 0.001 M in dithiothreitol rather than 0.01 M in 2-mercaptoethanol. (The term "buffer" refers to 0.05 M potassium phosphate buffer, pH 7.0.) The enzyme purification was revised beginning with the hydroxyapatite fractionation step [3]. At this point the Sephadex G-100 eluate was diluted 6-fold with 0.001 M dithiothreitol and applied to a 6 cm × 3.7 cm column of hydroxyapatite (Biogel HT, BioRad Laboratories, Richmond, CA) equilibrated with 0.01 M potassium phosphate buffer, pH 7.0, and packed on top of a 1.0 cm layer of Sephadex G-25. The column was washed with 400 ml of 0.01 M potassium phosphate, pH 7.0, prior to application of the enzyme solution and was eluted with a linear gradient obtained with 800 ml of 0.25 M potassium phosphate buffer, pH 7.0, in the reservoir and an equal volume of 0.01 M potassium phosphate buffer, pH 7.0, in the mixing vessel. A flow rate of 50 ml per h was maintained with a 2 m pressure head. A typical elution profile is shown in Fig. 1. Solid ammonium sulfate was added to the pooled active fractions to 65 ~ saturation and the precipitate was collected and dissolved in 3.0 ml of buffer 5 mM in UDPglucose and stored at --70 °C (hydroxyapatite fraction). The hydroxyapatite fraction was dialyzed against three 2 liter volumes of buffer for a period of 6 h before chromatography on a 1.5 cm × I0 cm column of diethyl-

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Fig. 1. Elution profiles of UDPglucose dehydrogenase from columns of (a) hydroxyapatite and (b) diethylaminoethane Sephadex.

aminoethane (DEAE) Sephadex A-50. The column was washed with 100 ml of buffer before application of the enzyme solution, and eluted with a linear gradient obtained with 115 ml of 0.2 M potassium phosphate, p H 7.0, in the reservoir and 115 ml of buffer in the mixing chamber. A flow rate of 15 ml/h was obtained with a 0.6 meter pressure head. A typical elution profile from this column is shown in Fig. 1. Active fractions were concentrated with a m m o n i u m sulfate as described and dissolved in a minimal volume of buffer 5 m M in UDPglucose (DEAE-Sephadex fraction). RESULTS

Enzyme purification. As can be noted in Table I, an overall purification of 330-fold was achieved with recovery of 27 % of the activity in the crude extract. TABLEI P U R I F I C A T I O N OF U D P - G L U C O S E D E H Y D R O G E N A S E F R O M E. COL1

Crude extract (NH4)2SO4 fraction DEAE-cellulose fraction Sephadex G-100 eluate Hydroxyapatite fraction DEAE-Sephadex fraction

Volume (ml)

Total activity (units)

Total protein (rag)

Specific activity (units/mg)

Yield (~)

710 75 22 6.5 3.0 3.0

675 540 368 340 290 185

16 600 4 750 460 119 26 14

0.04 0.11 0.80 2.9 11,2 13,2

100 80 55 50 43 27

422

Enzyme stability. The hydroxyapatite fraction was stable for at least 4 weeks when stored frozen at --70 °C in buffer which was 5 mM in UDPglucose and 1 mM in dithiothreitol; often such storage resulted in a 50°i; increase in enzyme activity. However, the concentrated DEAE-Sephadex fraction was far less stable; storage under identical conditions caused 50 ~, loss of activity after one week. Homogeneity. The purified enzyme appeared to be monodisperse since upon ultracentrifugation it showed only one symmetrical peak, s20,w = 4.87 S. Upon polyacrylamide gel electrophoresis, multiple bands were obtained, even in the presence of dithiothreitol, probably because of the formation of oligomers. However, when tile enzyme was examined by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate after treatment with 2-mercaptoethanol and sodium dodecyl sulfate, a single symmetrical band was obtained (Fig. 2). In addition, treatment of the enzyme with 6.0 M guanidinium chloride yielded a product which was apparently monodisperse when examined in the ultracentrifuge, since a linear plot resulted when In concentration was plotted against l/r 2. The molecular weight of the subunits calculated from the ultracentrifugal data, using i~ calculated from the amino acid analysis and corrected for guanidinium chloride [7] was 41 000. Further indication of homogeneity of the dehydrogenase was the presence of only one N-terminal amino acid

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Fig. 2. Determination of the molecular weight of the subunits of UDPglucose dehydrogenase from E. coli by dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was mixed with three reference proteins and the mixture subjected to electrophoresis. The stained gel was scanned at 550 nm with a Gilford gel scanner. The peaks from left to right represent bovine serum albumin, UDPglucose dehydrogenase, chymotrypsinogen A, and egg white lysozyme, q-he insert is a plot of the logarithms of the molecular weights of the reference proteins versus their migration distance. The arrow indicates the migration distance of the dehydrogenase subunits.

423 in the purified enzyme. The results of peptide m a p p i n g are also consistent with the presence o f a h o m o g e n e o u s product. Amino acid analysis. The a m i n o acid c o m p o s i t i o n of the enzyme is presented in Table II. It is n o t e w o r t h y that there is n o detectable t r y p t o p h a n . The a m i n o acid c o m p o s i t i o n is presented as the nearest integer a s s u m i n g that the enzyme consists of identical polypeptide chains of molecular weight 47 000. This value was o b t a i n e d by statistical d e t e r m i n a t i o n [13] of the m i n i m a l molecular weight which would yield integral n u m b e r s of a m i n o acids. TABLE II AMINO ACID COMPOSITION OF UDP-GLUCOSE DEHYDROGENASE Amino acid

/~g amino acid residue/ mg protein Hydrolysis time (h) 24

Lysine Histidine Arginine Aspartate/asparagine Threonine Serine Glutamate/glutamine Proline Glycine Alanine l/2-Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan ~

48

Mean value

Number of residues/ 47 000 daltonsd

Integral number of residues/ 47 000 daltons

49.09 11.74 51.72 128.03a 25.11 31.27 b 99.50 a 27.45 21.95 42.92 ~ 3.39 51.27 ~ 18.21 77.94a 67.20b 37.24 43.19 a .

22,84 5.10 19.75 66.76 14.82 21.41 45.97 16.86 22.93 36.00 1.96 30.86 8.28 41.07 35.91 13.61 17.5

23 5 20 67 15 21 46 17 23 36 2 31 8 41 35 14 18 0

96

51.02 4 2 . 7 2 53.54 12.60 11.72 10.91 60.73 4 8 . 0 6 46.35 98.58 109.04 128.03 24.24 2 4 . 7 4 26.35 31.06 3 1 . 4 8 27.88 7 7 . 9 9 8 8 . 0 3 99.50 -26.19 28.70 -22.35 21.55 35.25 4 0 . 6 4 42.92 -3.16 3.62 40.95 4 6 . 2 7 51.27 18.95 17.98 17.71 50.73 6 4 . 8 3 77.94 53.71 6 3 . 9 8 67.20 36.35 3 8 . 0 3 37.34 36.57 3 8 . 8 5 43.19 . . . .

a Value reported is for 96 h hydrolysis. b Value is extrapolated to zero time. c Determined by the method of Edelhoch [10]. d Calculated according to Delaage [13].

Ultraviolet spectrum. The enzyme gave a typical protein a b s o r p t i o n spectrum with a single a b s o r p t i o n m a x i m u m at 277 n m and a slight shoulder at 281 nm. A s o l u t i o n c o n t a i n i n g 1 m g of enzyme/ml (determined according to the m e t h o d of Lowry et al. [5]) gave an a b s o r b a n c e of 0.39 at 277 nm. The a b s o r p t i o n ratio 280 n m / 260 n m was 1.80. Polyaerylamide gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme revealed a single symmetrical b a n d when examined by scanning of the stained gel at 550 nm. F r o m c o m p a r i s o n with proteins of k n o w n m o l e c u l a r weight a n d s u b u n i t c o m p o s i t i o n (Fig. 2), the molecular weight of the polypeptide chain is determined to be 47 i300, in excellent agreement with the value obtained by statistical analysis of a m i n o acid c o m p o s i t i o n .

424 N-terminal amino acid analysis. The only major dansyl amino acid detected in the protein was dansyl arginine. Minor fluorescent spots corresponding to dansyl-Otyrosine and dansyl hydroxide were also present as was a minor unidentified component near dansyl arginine. L-Arginine is therefore the sole amino terminal amino acid present in UDPglucose dehydrogenase from E. coli. Peptide "map". The peptide map contained 44 peptides; no ninhydrin staining material remained at the origin. DISCUSSION The preparation described yields a homogeneous protein, as judged by the presence of a single molecular species upon ultracentrifugation of the intact enzyme and of the subunits produced by treatment with 6.0 M guanidinium chloride. In addition, a single molecular species was obtained upon examination of the subunits in dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of only one amino terminal amino acid is also in accord with enzyme homogeneity. The peptide map of the UDPglucose dehydrogenase contains 44 peptides, which apparently represents the totality of the material resulting from the tryptic digestion, since no ninhydrin-staining compounds remained at the origin of the electropherogram. The enzyme contains 43 arginine plus lysine residues/47 1300 molecular weight, which is consistent with identity of the subunits, as is the presence of a single amino terminal amino acid. The molecular weight of the subunit of UDPglucose dehydrogenase is 47 000, as determined both by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by amino acid analysis. The subunit molecular weight calculated from the results of ultracentrifugation in 6 M guanidinium chloride is somewhat smaller, 41 0130. The discrepancy probably represents inadequate correction for ~ provided by the method of Hade and Tanford [7]. The molecular weight of the intact enzyme has been shown by gel filtration to be in the order of 86 000 [3]. From all the data presented it seems probable that the UDPglucose dehydrogenase is constituted of two identical or very similar subunits of molecular weight 47 000. E. coli UDPglucose dehydrogenase differs from the bovine liver enzyme in respect to structure as well as in its kinetic properties. The bovine enzyme consists of six identical subunits of molecular weight 52 000, has a high thiol content (12/subunit [1, 2]), and contains approximately six tryptophan residues/subunit. The E. coli enzyme, on the other hand, consists of two identical subunits of molecular weight approximately 47 000, has a low cysteine content (2 residues/subunit), and is completely devoid of tryptophan. The turnover number of the bacterial enzyme is 4-fold greater than that of bovine UDPglucose dehydrogenase, and as has been mentioned previously [3], the bacterial enzyme is not subject to the particular type of positive cooperative inhibition by UDP-D-xylose seen with the enzyme from beef liver as well as from other eukaryotic sources. The higher molecular weight and possibly greater structural complexity of the bovine liver enzyme may reflect its more complex control requirements. In eukaryots, UDP-D-glucuronate serves not only as a source of the D-glucuronosyl moiety but also as a precursor of the D-xylosyl donor UDP-D-xylose; it has been suggested that the latter is a specific feedback inhibitor of the UDPglucose dehydrogenase in such organisms. The probable absence of D-xylose (and also of

425 UDP-D-xylose) f r o m the c o m p l e x saccharides o f the E. coli strain used in this s t u d y is consistent with the presence o f a smaller, simpler U D P g l u c o s e d e h y d r o g e n a s e which lacks the s t r u c t u r a l c o m p l e x i t y needed for the c o n t r o l aspects of the beef liver protein. ACKNOWLEDGMENTS This research was s u p p o r t e d by a g r a n t ( A M 15322) f r o m the U n i t e d States Public H e a l t h Service. One o f the a u t h o r s ( D . S . F . ) was a R e s e a r c h C a r e e r D e v e l o p m e n t A w a r d ( 1 - K 3 - G M 28296) g r a n t e e o f the N a t i o n a l Institutes of Health, U n i t e d States Public H e a l t h Service. The a u t h o r s w o u l d like to t h a n k Dr. C. Coffee and Ms. I. K u o for p e r f o r m i n g the s e d i m e n t a t i o n velocity experiments. REFERENCES 1 Gainey, P. A., Pestell, T. C. and Phelps, C. F. (1972) Biochem. J. 129, 821-830 2 Uram, M., Bowser, A. M., Feingold, D. S. and Lamy, F. (1972) Anal. Asoc. Chim. Argent. 60, 135-140 3 Schiller, J. G., Bowser, A. M. and Feingold, D. S. (1973) Biochim. Biophys. Acta 293, 1-10 4 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 5 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 6 Yphantis, D. (1964) Biochemistry 3, 297-317 7 Kirby-Hade, E. P. and Tanford, C. (1967) J. Am. Chem. Soc. 89, 5034-5040 8 Crestfield, A. M., Moore, S. and Stein, W. H. (1973) J. Biol. Chem. 238, 622-627 9 Beale, D. (1969) in: Chromatographic and Electrophoretic Techniques (Smith, I., William Heinemann Medical Books, Ltd., London 10 Edelhoch, H. (1967) Biochemistry 6, 1948-1954 11 Gray, W. R. (1972) Methods Enzymol., Vol. XXV, Part B, 121-138 12 Woods, K. R. and Wang, K. (1967) Biochim. Biophys. Acta 133, 369-370 13 Delaage, M. (1968) Biochim. Biophys. Acta 168, 573-575