PROTEINS Structure, Function, and Genetics 9:135-142 (1991)

Preliminary Crystallographic Results

The Isolation, Purification, and Preliminary Crystallographic Characterization of UDP-Galactose-4-Epimerase From Escherichia coZi Alan J. Bauer,'92Ivan Rayment,lV2 Perry A. Frey,lV2 and Hazel M. Holden'*3 'Institute for Enzyme Research, Graduate School, the 2Department of Biochemistry, College of Agriculture and Life Sciences, and 3Department of Chemistry, College of Letters and Science, University of Wisconsin-Madison, Madison, Wisconsin, 53705

ABSTRACT Uridine diphosphogalactose4-epimerase from E. coli has been crystallized in a form suitable for a high-resolution X-ray crystallographic structural analysis. The enzyme complexed with a substrate analogue, uridine diphosphobenzene (UDP-benzene), crystallizes readily using polyethylene glycol goo0 as the precipitant. The crystals belong to the orthorhombic space group P2,2,2, with unit cell dimensions,a = 76.3 hi, b = 83.1 hi, and c = 132.1 hi. Based on still setting photographs, the crystals diffract to a nominal resolution of 2.3 hi and are stable in the X-ray beam. The enzyme used in these experiments was produced by a new expression system and a modified purification scheme. Key words: X-ray crystallography, galactose metabolism, nucleotide binding, nonstereospecific hydride transfer, protein structure INTRODUCTION Uridine diphosphogalactose-4-epimerase (EC 5.1.3.2) carries out the interconversion of UDP-galactose and UDP-glucose (Fig. 1). The enzyme from E . coli is a dimer of identical subunits and has a molecular weight of 79,000.' One molecule of nicotinamide adenine dinucleotide (NAD+) is noncovalently bound to each dime? and has been implicated in the transient oxidatiodreduction of the sugar moeity of the Isotope conservation a t C-4 of the sugar: the absence of isotope exchange between solvent and s u b ~ t r a t e ,and ~ the chemical competence of 4-keto 6-deoxyglucoseUDP' suggest that a reduced enzyme-bound nucleotide and a 4-ketopyranose intermediate are formed during epimerization. Return of the hydride from NADH to the opposite face of the ketone intermediate results in product formation. There are marked differences in the relative binding affinities of the uridine nucleotide and sugar 0 1991 WILEY-LISS, INC.

m o i e t i e ~While .~ binding of UDP shows a net free energy change on the order of -7 kcal/mol, the binding of the hexopyranose is relatively nonspecific. A model proposed by Freyg to explain the action of the epimerase suggests that the energy introduced by enzyme-uridine nucleotide interaction is used to effect a conformational change which increases the reactivity of NAD+. After the hydride is transferred from the C-4 position of the substrate to the pyridine nucleotide, there is rotation about the phosphorousglycosyl oxygen bond to allow the return of the same hydride to the opposite face of the ketone intermediate (Fig. 2). This model for epimerization suggests that the enzyme can assume different conformations in the presence and absence of uridine nucleotides. Conversion of the free enzyme into its active conformation occurs upon binding of the uridine nucleotide portion of the substrate. This model demands a minimum of two distinct conformations of the epimerase but does not preclude the existence of still other forms of the enzyme. There are several interesting structural issues raised by this model of epimerase action. For example, how does the enzyme facilitate the nonstereospecific hydride transfer between P-NADH on the enzyme'' and the 4keto hexopyranose intermediate? Is the intermediate free to rotate within the active site? Which base removes the C-4 hydroxyl proton to initiate catalysis, and how does this base act on the hydroxyl protons of both glucose and galactose? How do identical subunits noncovalently bind a single molecule of NAD ? We report here the subcloning and overexpression of the epimerase gene, as well as the purification, crystallization, and preliminary crystallographic characterization of the enzyme. Though the epimerase crystallized in the presence of several different +

Received June 29, 1990; accepted July 16, 1990. Address reprint requests to Hazel M. Holden, Institute for Enzyme Research, The University of Wisconsin-Madison,1710 University Ave., Madison, WI 53705.

136

A.J. BAUER ET AL.

Protein Chemistry

H

HO .-

H o g 0

0

0-UDP

(

0-UDP

Fig. 1. Reaction catalyzed by UDP-galactose-4-epirnerase. Reprinted from F r e ~ . ~

precipitants, only one crystal form has proved suitable for a high-resolution structural analysis.

MATERIALS AND METHODS Chemicals and Enzymes All restriction enzymes, T4 DNA ligase, and appropriate salts were purchased from BoehringerMannheim Biochemicals (Indianapolis, IN) and used according to the specifications of the manufacturer. Galactose, ampicillin, streptomycin sulfate, NAD+ , UDP-glucose dehydrogenase, phenylmethylsulfonyl flouride, bicine (free acid), tetrasodium EDTA, PIPES (free acid), succinic acid, citric acid, and EPPS (free acid) were purchased from Sigma Chemical Co. (St. Louis, MO). Bacto tryptone and bacto yeast were purchased from Difco Labs (Detroit, MI). Ammonium sulfate (enzyme grade) was purchased from Schwarzi Mann Biotech (Cleveland, OH). Sodium phosphate (mono- and dibasic) and potassium phosphate (monoand dibasic) were purchased from Fisher Chemical Co. (Fairlawn, NJ). Polyethylene glycol 8000 (PEG 8000) was purchased from Baker Chemical Co. (Phillipsburg, NJ). Sodium sulfate was purchased from EM Science (Cherry Hill, NJ). Uridine diphosphobenzene (UDP-benzene) was the generous gift of Dr. George Flentke.

Chromatography gephadex DEAE A-50-120 (Sigma) and hydroxyapatite (Bio-Rad, Richmond, CA) resins were prepared, washed with 20 mM potassium phosphate buffer, and run according to the specifications of the manufacturer.

Electrophoresis All protein electrophoresis was carried out on a Phastgel (Pharmacia, Piscataway, N J ) system with preformed 4.3 cm x 5 cm gels. Native (8-25% acrylamide), SDS (12.5% acrylamide), and isoelectric f o c u ~ i n ggels ~ ' ~were ~ ~ run according to the programs supplied by the manufacturer. All DNA electrophoresis was carried out on 1% agarose gels. Standard agarose (molecular biology grade agarose, IBI, New Haven, CT) electrophoresis was performed on minigels." Low-melting point agarose (Sea Plaque, Marine Colloids, Rockland, ME) electrophoresis was performed on minigel plates as per the method of Struhl."

Protein solutions were concentrated in a n Amicon ultrafiltration cell with YM Diaflo membranes. Amicon Centriprep (5-15 ml, M , = 10,000) and Centricon (2-5 ml, M , = 10,000) concentrators were used for concentrating smaller protein solutions. Dialysis tubing (25 mm diameter, M , = 12,000) was boiled for 3 min in dilute EDTA and sodium bicarbonate just prior to use. The concentration of enzyme was based upon A,,,,'.'" = 1.05l" for a 1 cm light path.

Bacterial Strains

E. coli strains JMlOl and BL21 (DE3), pI24 were the generous gift of Dr. Teresa Field. Plasmid pI24'" is a construct of bacterial vector pBR32211 which contains the entire gal operon. BL21 (DE3), pLysS was the generous gift of Mr. Stewart Loh. The BL23 (DE3) strain of E. coli is a construct of Studier'" and contains a T7 RNA polymerase behind a lacUV5 promoter in the host genomic DNA. The pLysS plasmid codes for a small lysozyme, and the presence of this plasmid tends to decrease t h e loss of construct plasmid during cell growth.'" JMlOl pTZ18R ( a modified version of pBR322 which contains a T7 RNA polymerase promoter just upstream of a multicloning site situated in the lac Z gene) was purchased from U.S. Biochemicals (Cleveland, OH). Media Rich medium is supplemented 2YT' I and contains in 1 liter bacto tryptone (16 g), bacto yeast (10 g), and NaCl (5 g). H-plates" were made from a solution which contains (in 1 liter) agarose (12 g), bacto tryptone (10 g), and N a C l ( 8 g). All media were made 100 pgiml in ampicillin just prior to use.

Temperature All experimental manipulations were performed at 4°C except where otherwise noted. All enzymatic assays were performed at 25°C.

Instruments Assays of UDP-galactose-4-epimerase activity were carried out on either of the following spectrophotometers: a Cary 118 or a Hewlett-Packard 8452A Diode-Array Spectrophotometer. Determinations of protein concentrations were carried out on a Beckman DU spectrophotometer. pH was measured on a Beckman SS-1 pH meter.

Assays Activity of the epimerase was monitored using the coupled assay described by Hogness and Wilson.'

Subcloning of the Epimerase Gene The gene for the epimerase and the gal promoter region were moved from plasmid pI24 to a position on vector pTZ18R downstream of the T7 promoter.

CRYSTALLIZATION OF UDP-GALACTOSE-4-EPIMERASE HO

,t..d"J&, no

0-UOP

-

~ N A O HO=&)

'uop-cfl E-NAO~

H K

137

HO

olo-lloP

11

HO

*"op-G,&

E ~ N A O ~

OH

0-UOP

E%AOH

Fig. 2. Possible mechanism for epimerase action. Reprinted from F r e ~ . ~

Large amounts of plasmid and vector DNA were isolated by a standard miniprep procedure." Plasmid DNA from BL21 (DE3), pI24 was incubated with restriction endonucleases EcoRI and Puu I1 overnight a t 37°C. The T7 vector DNA from JMlOl pTZ18R was incubated with EcoRI and Hind111 under identical conditions. The linear vector, as well as the target 1500 base-pair insert were visualized on a 1%agarose gel. Two micrograms of restricted DNA were run on a low-melting point agarose gel and the appropriate bands were excised, combined in a ratio of two to one (insert to vector) and ligated overnight as per the method of Struhl.12The ligated DNA was serially diluted and introduced into competent" E. coli JMlOl cells. Successful transformants were selected via a bluelwhite screen on H plates which were 47 Fg/ml of IPTG (to increase expression of the gene behind the T7 promoter) and 40 Fglml of Xgal.l1 Successful transformants were also identified by high epimerase activity in crude lysate samples of overnight growths. The construct plasmid, pT7E, was isolated from the transformed JMlOl cells by the same miniprep technique employed earlier. Restriction of the plasmid DNA with EcoRi and HindIII produced the fragments expected from a successful clone. The construct DNA was again serially diluted and added to competent BL21 (DE3), pLysS cells. Transformants were selected from H-plates and grown overnight in 5 ml of rich medium. The cells were made 15% in glycerol, quick frozen in liquid nitrogen, and stored at -70°C. These cells will be referred to as E. coli BL21 (DE3), pLysS, pT7E. Expression of the Epimerase

E. coli BL21 (DE31, pLysS, pT7E cells were streaked out on H plates and incubated at 37°C overnight. A single colony was used to inoculate 10 ml of rich medium, which was shaken a t 37°C overnight. When the starter culture reached an optical absorbance density, Asoo, of 1.0 (approximately 16 hr), this growth was used to inoculate (0.2%)six 2 liter shaker flasks (500 ml rich medium per flask). The

large-scale growth was shaken at 37°C for 4 hr, a t which time additional ampicillin (50 mg per flask, added as a sterile solution) was added. The growth was allowed to continue for 7 more hours, at which time the cells were harvested by centrifugation (2661g for 10 min), quick-frozen in liquid nitrogen, and stored a t -70°C. The yield of cells was 24 g of frozen cells from 3 liters of growth. The purification scheme of the epimerase is a modified protocol of Wilson and Hogness.' The cells were thawed and resuspended in 50 ml of 20 mM monobasic potassium phosphate, titrated to pH 7.2 with 10 M KOH. [Note: all buffers in the purification scheme contained 1mM tetrasodium EDTA and 1 mM phenylmethylsulfonyl flouride.] The pLysS plasmid codes for a lysozyme which effectively served to lyse the cells during a 40 min resuspension. Trace amounts of deoxyribonuclease and CaC1, were added to reduce the viscosity of the solution. The lysate was spun a t 10,240gfor 30 min to remove cell debris. Streptomycin sulfate (10%)(0.254 mum1 of solution) was slowly added to the supernatant. After 20 min of additional stirring, the solution was centrifuged a t 10,240g for 20 min. Finely ground ammonium sulfate was slowly added to the resulting supernatant to make a solution 45% in ammonium sulfate. This solution was allowed to stir for an additional 30 min and was then centrifuged at 10,240g for 20 min. The pellet was resuspended in the phosphate buffer (15 ml) and dialyzed against the same buffer (2 liters) for 18 hr. This material was carefully loaded onto a 500 ml hydroxyapatite column (24 cm2 x 30 cm) which had been preequilibrated with 20 mM potassium phosphate, pH 7.2. The column was eluted with the same buffer and fractions (10 ml) which showed epimerase activity were analyzed by native gel electrophoresis. Those fractions which contained epimerase (20-50) were pooled and carefully titrated to pH 8.5 with dilute KOH. This protein solution was then loaded onto a 350 ml DEAE A-50-120 column (7 cm2 x 60 cm) which had been preequilibrated with 20 mM dibasic

138

A.J. BAUER ET AL.

phosphate titrated to pH 8.5 with 1 M monobasic potassium phosphate. After the sample (300 ml) had loaded, the protein was eluted with a linear gradient, 20-300 mM potassium phosphate, pH 8.5 ( 2 x 800 ml). Fractions (10 ml) were assayed for enzyme activity and further analyzed by native gel electrophoresis. Fractions (100-125) containing significant epimerase activity which showed a single band on a native gel were pooled and concentrated from 300 to 30 ml via a n Amicon concentrator. The epimerase was further concentrated using Centriprep and Centricon concentrators to concentrations ranging from 10 to 136 mgiml.

Crystallization of the Epimerase Crystallization conditions were surveyed using the hanging drop method of vapor d i f f u ~ i o n ' a~t room temperature and at 4°C. Epimerase samples were buffered in 20 mM potassium phosphate, 5 mM sodium azide, pH 7 . 5 . For X-ray diffraction experiments, the crystals were mounted in thin-walled quartz capillary tubes. Precession photographs were recorded using nickel-filtered copper K , radiation from a Rigaku RU 200 rotating anode X-ray generator operated at 50 kV and 50 mA with a 200 pm focal cup. The incident beam was focused with a set of nickel-coated double focusing mirrors. The exposure time was typically 20 h r for a 13"precession photograph at a crystal-to-film distance of 100 mm.

Heavy Atom Derivative Searches Large single crystals of the epimerase complexed with UDP-benzene were transferred from the crystallization media to a synthetic mother liquor containing 10% PEG 8000, 200 mM NaCl, 2 mM UDPbenzene, 50 mM sodium succinate, pH 6.0. The crystals were next transferred to a 13% PEG solution with 200 mM NaCl, 2 mM UDP-benzene, 50 mM sodium PIPES, pH 7.0. Solutions of heavy atom compounds were made in this mother liquor at pH 7.0, and epimerase crystals were soaked for varying lengths of time. Potential derivatives were identified by precession photography. Three-dimensional X-ray data sets were collected using a Siemens X1000 Area Detector.

RESULTS The gene coding for the epimerase (galE)was successfully cloned into the T7 expression vector (Fig. 3). The presence of t h e gal promoter just upstream of the transcriptional start site allowed for significant basal expression of the epimerase gene in the absence of T7 RNA polymerase induction. Large growths (3 liters) were performed in the presence of ampicillin only. The final yield of epimerase (after purification) was generally between 125 and 175 mg/liter of culture media. The purification of the epimerase is summarized in Table I. The final specific activity of the enzyme

Fig. 3. Construct plasrnid. pT7E

was similar to published values for the epimerase isolated from E. coli regulatory mutant K12 gal ' (hdg).' SDS polyacrylamide gel electrophoresis showed a single band with subunit molecular weight of 39,000 Da. In attempts to crystallize the epimerase, the following precipitants were employed: polyethylene glycol 8000, ammonium sulfate, sodiumipotassium phosphate, sodium citrate, magnesium sulfate, and sodium sulfate. Initial experiments were performed with the enzyme in the absence of uridine nucleotides. Several crystal forms were obtained but none proved suitable for a detailed structural study. Data concerning these crystal forms are summarized in Table 11. Since 31PNMR data strongly suggested that the epimerase adopts a different conformation in the presence of uridine nucleotides,20 further experiments in the crystallization survey were performed in the presence of various substrate analogues. A new crystal form was grown using the enzyme complexed with uridine diphosphobenzene (UDPbenzene). These crystals were obtained from a solution containing 6.5% PEG 8000, 200 mM NaCl, 2 mM UDP-benzene, 5 mM NaN,, buffered in 50 mM sodium succinate, pH 6.0 (4°C).The enzyme concentration was 30 mgiml. Large single crystals ( 2 m m ) were grown by the batch method," using macroseeding techniques."" Crystal growth was generally complete in 5 weeks. The crystals of the enzyme complexed with UDP-benzene are stable in the Xray beam and are suitable for a high resolution Xray diffraction analysis. Figures 4 and 5 show 13" precession photographs of the hkO and Okl zones. Results of three-dimensional data collection are summarized in Table 111. An important observation concerning crystallization of the enzyme was made during the hanging-

CRYSTALLIZATION OF UDP-GALACTOSE-4-EPIMEFtASE

139

crystals grew in PEG solutions containing any one of the following salts: NaCl, KCl, Na,SO,, (CH,),NCl, NaNO,, and Na,EDTA. The concentration of PEG necessary for crystal formation decreased with increasing ionic strength. Figures 6 and 7 show typical epimerase crystals grown from solutions containing PEG 8000 and 200 mM KC1 (Fig. 6 ) or 200 mM NaCl (Fig. 7).

Fig. 4. Thirteen precession photograph of the Okl zone. The X-ray photograph was recorded as described in the experimental section. Diffraction maxima at the edge of the precession circle correspond to a resolution of 3.4 A.

Fig. 5. Thirteen precession photograph of the hkO zone. The X-ray photograph was recorded as described in the experimental section. Diffraction maxima at the edge of the precession circle correspond to a resolution of 3.4 A.

drop survey. When PEG 8000 was the precipitant, the epimerase would only crystallize in solutions containing several hundred millimolar salt. Since a solution containing polyethylene glycol could not balance the large excess charge on the epimerase (pZ = 5),23 the salts may have aided in the enzymeenzyme interactions essential for nucleation and crystal growth. Though the presence of salt was absolutely necessary for crystal formation, the specific salt employed seemed less important. Epimerase

CONCLUSIONS In overexpressing the gene for the epimerase, the intention was to remove several of the steps from the original purification protocol. After successful subcloning of the gene, it was realized that blunt and sticky ends had to ligate in order to seal one end of the construct. The presence of large amounts of epimerase activity in the crude lysate of overnight growths, as well as the loss of one Hind111 sight on the construct plasmid, strongly suggested that such a ligation phenomenon actually did occur. Since the cloning strategy also moved the gal promoter into the plasmid, there was significant basal expression of the epimerase gene in the absence of any induced T7 RNA polymerase production. Isopropyl-P-thiogalactopyranoside (IPTG), a gratuitous inducer of the lacUV5 promoter, was thus not added so as to avoid sugar-induced reduction of the epimera~e.~, Typically, 5% of the epimerase isolated during a preparation is inactive and is thought to carry the reduced pyridine n~cleotide.'~ The purification of the epimerase followed along the lines of earlier protocols. During the first day of the new preparation procedure, however, an alkaline ammonium sulfate (pH 10.5) and several 4 M dibasic potassium phosphate precipitation steps were eliminated because these steps can lead to microheterogeneity in the enzyme sample. The hydroxyapatite column was retained as it effected a 4-fold purification of the enzyme (Table I). The anion-exchange column allowed for the separation of the three remaining major protein constitutents. Activity assays and acrylamide gel profiles show that the final sample of epimerase is better than 95% pure and of high specific activity. All epimerase crystallization experiments involved the enzyme obtained from the modified isolation and purification scheme. Initial experiments were performed on the native enzyme free of any uridine nucleotides. The first promising epimerase crystals were grown from a solution of sodiudpotassium phosphate (Table 11).Though large crystals could readily be grown by the sitting drop method,17 the large unit cell and poor diffraction qualities of these crystals made them unsuitable for a detailed structural study. After additional hanging drop experiments were performed, a second crystal form was isolated from PEG 8000 (Table 11).Though these crystals diffracted to 3.0 A and showed a relatively low mosaic spread, there were eight epimerase

140

A.J. BAUER ET AL.

TABLE I. Purification of t h e Epimerase Isolated From a 3 liter Growth of E . coli BL21 (DE3), pLysS, pT7E

Step Crude lysate (NH, )?SO, precipitation Hydroxyapatite column DEAE-A-50-120 column

mg of protein 12,000 3,500

Total units* 140,000 104,800

Specific activity (Uirng) 11.7 29.9

X-fold purification 1.0 2.6

Yield (%)

100 75

93,200

800

116.5

10.0

67

87,500

375

233.3

19.9

62

'One unit of epimerase activity is defined a5 the conversion of 1 p n o l of UDP-galactose to UDP-glucose per minute

TABLE 11. Various Crystal Forms of UDP-Galactose-4-Epimerase

Crystallization conditions 0.7 M Na / K . phosphate, 50 mM EPPS, pH 8.0, 1%(viv) PEG 400, 5 mM NaN:,, 30 mgiml epimerase, 4°C 0.65 M Na,SO,, 50 mM sodium bicine, pH 8.5, 5 mM NaN:,, 136 mgiml epimerase. room temperature 12% (wiv) PEG 8000, 50 mM sodium PIPES, pH 7.0, 200 mM KCl, 5 mM NaN:,, 30 mgiml epimerase, +

Space group c2

Dimers/A.U.* 8

c2

8

Unit cell dimensions a = 194.6 A b = 86.7 Ac = 305.3 A @ = 99.2"

Maximum resolution of diffraction (A) 3.5

148.2 A 89.5 A = 348.0 A B = 92.0"

3.0

76.3 A 83.1 A 132.1 A

2.3

a b c

= =

a

= = =

4°C 6.5% (w/v)PEG 8000, 50 mM sodium succinate, pH 6.0, 200 mM NaCI, 1.9 mM UDP-benzene, 5 mM NaN:,, 30 mgiml epimerase,

.

L

b c

4°C

'The number of dimers in the asymmetric unit is based on the solvent parameter V, observed value for globular proteins.'" :This crystal form has not yet been characterized.

dimers in the asymmetric unit. It was observed, however, that when these latter crystals were soaked in a synthetic mother liquor containing 1 mM UDP-benzene, the diamond shaped crystals dissolved and rod-shaped crystals appeared. Consequently, further crystallization trials were conducted using PEG 8000 with UDP-benzene and large crystals were subsequently obtained. These crystals belong t o the orthorhombic space group, P2,2,2, with unit cell dimensions a = 76.3 A, b = 83.1 A, and c = 132.1A. The crystals a r e stable in the X-ray beam and are easy to manipulate. There is one epimerase dimer in the asymmetric unit, and the volumeiunit molecular weight in the unit cell (V,) is

=

2.15 A:'/Da, which is the most commonly

2.6 A3/Da. This value lies within the normal range (1.68-3.53 A3/Da) observed for globular proteins. Crystals of the epimerase complexed with UDPbenzene diffract to a resolution of 2.3 A and are suitable for a high-resolution structural analysis. Threedimensional native crystal data have been collected to a nominal resolution of 2.7 A. Two heavy atom derivative data sets have also been collected, and the location of several heavy-atom binding sites have been determined by the analysis of Patterson maps. A search for other heavy-atom derivatives is in progress. In conclusion, to better understand the structural basis of nonstereospecific hydride transfer which oc-

141

CRYSTALLIZATION OF UDP-GALACTOSE-4-EPIMERASE

TABLE 111. Preliminary Area-Detector Data Summary Heavy-atom derivative Native K,PtCl, (CHAPbCHXOOH ....,

R,kt

Length of soak (davs)

Rint*

(%)

(%,

(A,

Number of heavy atoms bound

-

5.5 6.0 6.4

-

2.7 3.0 3.0

3 2

4 3

24 20

Resolution

*Rintis defined as SF-fllXZ. tRxa,e is defined as I/pNl-pHl /ZlF,I where pNIis the native structure factor amplitude and pH/ is the derivative structure factor amplitude.

Fig. 6. Crystals of free UDP-galactose-4-epimerasegrown using PEG 8000 as the precipitant.

Fig. 7. Crystals of UDP-galactose-4-epimerase complexed with UDP-benzene grown using PEG 8000 as the precipitant.

curs in the reaction catalyzed by UDP-galactose-4epimerase, crystals of the enzyme complexed with a substrate analogue have been grown. A detailed Xray study of these crystals will yield valuable information regarding the method of interaction between monomers and the nicotinamide coenzyme, as well as between the enzyme and substrate. Further structural studies of the free enzyme as well as of the reduced enzyme complexed with UDP-benzene will be necessary in order to understand on a molecular level how the enzyme uses the energy of bind-

ing of the uridine nucleotide to effect the oxidation/ reduction of the sugar moiety.

ACKNOWLEDGMENTS This work was supported by Grant GM30480 from the National Institute of General Medical Sciences, United States Public Health Service (to P.A.F.) and by National Institutes of Health Molecular Biology Training Grant GMO7215-14 (to A.J.B.).

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A.J. BAUER ET AL.

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Spring Harbor, New York Cold-Spring Harbor Laboratory Press, 1989. 12. Struhl, K. A rapid method of creating recombinant DNA molecules. Biotechniques 3(6):425, 1985. 13. Bradford, M. M. Spectrophotometeric method for the determination of protein concentrations. Anal. Biochem. 72: 248,1976. 14. Field, T. L. Galactose-1-phosphate uridylyltransferase: Identification of His 164 and His 166 as critical residues by site directed mutagenesis. Ph.D. Thesis, University of Wisconsin-Madison, 1988. 15. Studier, F. W., Moffatt, B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189113, 1986. 16. Moffat, B. A., Studier, F. W. Use of T7 lysozyme to improve the T7 gene expression system. Cell 49221, 1987. 17. McPherson, A. “Preparation and Analysis of Protein Crystals.” New York: Wiley-Interscience, 1982. 18. Phillips, W. C., Rayment, I. A systematic method for aligning double-focusing mirrors. Methods Enzymol. 114:316, 1985. 19. Matthews, B. W. Solvent content of protein crystals. J. Mol. Biol. 33:491, 1968. 20. Konopka, J . M., Halkides, C. J., Vanhooke, J. L., Gorenstein, D. G., Frey, P. A. UDP-galactose 4-epimerase. Phosphorous-31 nuclear magnetic resonance analysis of NAD+ and NADH bound a t the active site. Biochemistry 28:2645, 1989. 21. McPherson, A. Use of polyethylene glycol in the crystallization of macromolecules. Methods Enzymol. 114:120, 1985. 22. Thaller, C., Eichele, G., Weaver, L. H., Wilson, E., Karlsson, R., Jansonius, J. N. Seed enlargement and repeated seeding. Methods Enzymol. 114:128, 1985. 23. Unpublished data. 24. Kang, U. G., Nolan, L. D., Frey, P. A. Uridine diphosphate galactose-4-epimerase: Uridine monophosphate-dependent reduction by a- and p-D-glucose. J . Biol. Chem. 250: 7099,1975.