Biol. Chem. Hoppe-Seyler Vol. 373, 1067-1073, October 1992
Dihydropteridine Reductase from Escherichia coli Exhibits Dihydrofolate Reductase Activity Subhash G. VASUDEVAN*, Bela ?AALb and Wilfred L.F. ARMAREGO b Research School of Chemistry3 and Protein Biochemistry Group b , Division of Biochemistry and Molecular Biology, The John Curtin School of Medical Research, The Australian National University, Canberra City, Australia (Received 10 August 1992)*
Summary: E. coli Dihydropteridine reductase, known to have a pterin-independent oxidoreductase activity with potassium ferricyanide as electron donor, has now been shown to possess also dihydrofolate reductase activity. The kinetic parameters for dihydrofolate reductase activity have been determined. The ratio of the three activities, dihydropteridine reductase, dihydrofolate reductase and pterin-independent oxidoreductase activity is 1.0, 0.05 and 4.3, respectively. The enzyme, a flavoprotein which is unstable in the presence of dithiothreitol, was shown to be a
monomer with a molecular mass of 25.7 kDa. The apparent lack of discrimination between hydride transfer from the pyridine nucleotide to N5 of the pterin in the dihydropteridine reductase reaction and C6 of folate in the dihydrofolate reaction suggested that the FAD prosthetic group may be involved in the hydride transfers. The flavoprotein inhibitor N,N- dimethylpropargylamine inhibited the dihydropteridine reductase and oxidoreductase reactions differently and did not affect the dihydrofolate reductase activity however.
Key terms: Dihydropteridine reductase, dihydrofolate reductase, flavoprotein, hydride transfer, /V,yV-dimethylpropargylamine, E. coli.
Dihydropteridine reductase (EC. 1.6.99.7, DHPR) catalyses the reduction of "quinonoid" dihydrobiopterin. The latter is the natural cofactor for phenylalanine (EC 1.14.16.1)[1], tyrosine (EC 1.14.16.2)[2] and tryptophan (EC 1.14.16.4)[3] hydroxylases, and also for other enzymic reactions that require further
characterization e.g. glyceryl etherase (EC 1.14.16.5)^. A reductase from Escherichia coli with dihydropteridine reductase activity has been isolated to electrophoretic homogeneity. Unlike other dihydropteridine reductases that have been studied E. coli reductase possesses an FAD prosthetic
Enzymes : Dihydrofolate reductase, 5,6,7,8-tetrahydrofolate:NADP® oxidoreductase (EC 1.5.1.3); Dihydropteridine reductase, NAD(P)H:6,7-dihydropteridine oxidoreductase (EC 1.6.99.7); Glyceryl-ether monooxygenase, 1-alkyl-sn-glycerol, tetrahydropteridine:oxygen oxidoreductase (EC 1.14.16.5); Phenylalanine 4-monooxygenase, L-phenylalanine, tetrahydrobiopterin:oxygen oxidoreductase (4-hydroxylating) (EC 1.14.16.1) (in this paper named phenylalanine hydroxylase); Tryptophan 5-monooxygenase, L-tryptophan, tetrahydropteridine:oxygen oxidoreductase (5-hydroxylating) (EC 1.14.16.4) (in this paper named tryptophan hydroxylase); Tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating) (EC 1.14.16.2) (in this paper named tyrosine hydroxylase). Abbreviations: DHFR, dihydrofolate reductase; DHPR, dihydropteridine reductase; DTT, dithiothreitol; FPLC, fast-protein liquid chromatography; Hepes, 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide; Temed, Ν, Ν, Ν', Ν' -tetramethylethylenediamine. * Received by PTERIDINES 24 April 1992
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S. Vasudevan, B. Paal und W.L.F. Armarego
The superficial similarities between DHPR and dihydrofolate reductase (EC 1.5.1.3, DHFR) catalysed reactions (see Scheme 1) suggest that the two enzymes may have evolved from a common precursor and hence may share some structural similarities. Human liver cDNA[6J1 and a rat-liver cDNA[8] coding for DHPR have been cloned and sequenced. The amino-acid sequence deduced for the human-liver DHPR shows very little homology with that of human DHFR except for a short region around Cys104 for the former and Gly20 in the latter which is known to be important for pyridine nucleotide binding^. The extensive sequence identity between rat and human DHPR (only 10 conservative changes) implies that the structure of this enzyme is crucial for its function. In this paper we describe a purification procedure that gives a higher yield of the reductase from E. coli and present detailed evidence that the enzyme has DHFR activity. This is the first known enzyme to possess these two activities in the same protein. Part of this work was communicated at an international pteridine conference in Ziirich[9]. In contrast to our previous report the enzyme is functional as a protein of 25700 Da molecular mass and does not exist as a dimer. NADH,
NAD* H
H
NADP*
SCHEME 1
Materials and Methods E. coli K-12 derivative strains H712 and D3-157[10], and JFM 228'11' were used in this study. Purified E. coli DHFR and R-plasmid encoded DHFR were gifts from Dr. John F. Morrison (ANU). The 5,6,7,8-tetrahydropterins used in the assays were prepared by known procedures'12'. Chemicals of analytical or higher grades were used wherever possible. Growth of cells and preparation of cell-free extracts The media used and the method of growth for large scale enzyme preparations were as described'5'. Ampicillin (Beecham Research
Vol. 373 (1992)
Laboratories, Australia) was used at 50 μξ/ml for growth of JFM 228. The cells obtained after centrifugation were washed with 50mM Hepes/KOH pH 7.4 containing ImM EDTAand 200mM KCl and the cell-free extract was obtained as described previously'5'. Enzyme purification All manipulations were carried out at 4°C unless otherwise stated. The cell-free exract was fractionated with solid ammonium sulphate (84% of the DHPR activity was obtained in the 35-45% ammonium sulphate saturation) followed by centrifugation (150000xg). The precipitate was resuspended in 50mM Hepes/ KOH pH 7.4 (buffer A) and dialysed over-night against buffer A (3 x 2 /). The dialysate was applied onto a Fractogel TSK DEAE650 (M) column (2.0 x 15 cm, from Merck) which was equilibrated with buffer A. The column was washed with 10 column volumes of buffer A followed by elution with a linear gradient formed with 250 ml of buffer A and 250 ml of buffer A containing 200mM NaCl. The enzyme activity eluted at about lOOmM NaCl. The peak active fractions were pooled and the total protein was precipitated with ammonium sulphate (80% saturation) and pelleted by centrifugation. The pellet was re-suspended in buffer A and dialysed against it (3 x 500 ml). For long-term storage 20/iM NADH was added at this stage. When used immediately for the next purification step the pellet was dissolved in water (5 ml). The enzyme solution (2.5 m/) was loaded immediately on to two PD-10 columns (Sephadex G-25 pre-packed columns, Pharmacia) that have been pre-equilibrated with water. The combined eluates from two purifications (2x3.5 m/) of enzyme were made to 4% in ampholines (0.15 ml of Biolyte 3/10,40% w/v and 0.35 ml of Biolyte 5/7,40%; w/v from LKB) and applied to a preparative isoelectric focusing slab set up essentially as described by the manufacturer (Bio-Rad, Bio-Phoresis Instruction Manual). Bio-lyte electrofocusing gel (Bio-Rad 150 m/) made 4% in ampholytes (6 m/Biolyte 3/10,40% w/v and 9 m/Biolyte 5/7, 40% w/v) was set into a gel tray (20 x 11 x 0.8 cm) by wicking off the excess liquid from both ends of the tray. The sample was applied in two additions in the middle of the tray and wicking at the ends. The anode (0.33M H3PO4) and cathode (lM NaOH) were applied on thin-filter strips and placed at each end of the tray. Electrofocusing was carried out in Biophoresis cells (Bio-Rad) at constant voltage of 250 V for 2 h followed by 18 h at 700V. The yellow colour of the prosthetic group enabled easy visualization of the reductase which was closer to the anodic end. The yellow band was removed with a spatula, extracted with buffer A (3 x 5 ml) containing 200mM NaCl and centrifuged at 50000 x g min. The active functions were pooled and dialysed against buffer A (2 x 500 ml). The enzyme was stored in 1 ml aliquots with 20μΜ NADH at -20°C. The purification steps are summarised in Table 1. One activity unit (U) corresponds to μηιοί NADH oxidised x min""1 x (ml of enzyme)"1. Enzyme stability The purified protein (25 /xg, 50 μΐ) was incubated with various additives at 25 °C and -20°C (Table 2). The samples were thawed and 10 μΐ of sample was removed and assayed for DHPR activity'13' every 24 h. Dihydrofolate reductase assay The assay mixture contained: iMTris/HCl pH 7.4 (100 μ[), NADPH (ΙΟΟμΜ) and 7,8-dihydrofolate (ΙΟΟμ-Μ) in a total volume of 1 ml per cuvette at 25 °C. The reaction was initiated by addition of the enzyme (20 μΙ; 0.25 //.g of purified protein) into one cuvette. The initial rates were obtained from the rate of decrease of absorbance at 340 nm (ε for NAD(P)H is 620θΜ-1 cm"1). Note that the rate of decrease in absorbance is two times the real rate because 7,8-dihydrofolate absorbs at 340 nm with the same ε value. Thus a correction factor of 0.5 is used to obtain the rate of consumption of NADPH for the DHFR reaction. The kinetic parameters were obtained from the initial rates at various concentrations of one substrate and
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Vol. 373 (1992)
E. coli Dihydropteridine Reductase with Dihydrofolate Reductase activity
Table 1. Purification of DHPR from E. coli. Purification steps3 Volume
[m/] Cell-free extract Ammonium sulphate fraction 6 DEAEanion exchange0 Preparative isoelectric focusingd a
Total Total Spec. activity protein activity e [U] [U/mg]
Yield
[%]
100
303
5830
0.05
40
256
1350
0.19
84.5
26
210
142
1.9
63
9
120
27
4.5
100
40
Hepes KOH (pH 7.4) was used throughout the purification. A 35-45% ammonium sulphate fraction was used. DEAE-Fractogel. Using the Bio-Phoresis system (Βίο-Rad) with a pH gradient of4.5-7.5. For definition of U see Material and Methods.
b c d e
Table 2. Stability of DHPR from E. coli. Conditions3
ra DHPR without addition DHPR + DTT (2mM) DHPR + DTT (2mM) DHPR + NADH DHPR + NADH (20μΜ) DHPR + DTT(2mM) + NADH (20μΜ) DHPR -f DTT(2mM) + ΝΑΟΗ(20μΜ) a b c
DHPR activity [U]b after
Temp.
0.0 hc
24 h
48 h
72h
7.8
6.9
7.3
6.8
-20
5.3
3.7
2.8
2.3
25
5.3
1.2
0.3
0.0
-20
6.9
7.1
7.1
7.1
25
6.5
7.2
6.8
6.5
-20
5.8
5.7
5.4
4.7
25
5.8
5.3
4.6
3.8
20
In Hepes KOH (pH 7.4). For definition of U see Material and Methods. The activity at time = 0 h was carried out to correct for reduction of activity due to volume changes.
Table3. Dihydrofolate reductase activity of E. coli DHPR O.lM Tris/HCl,pH7.4at25°. Substrate
Km
Vmax3
[MM]
DHFA DHFA NADH NADPH a
27.3 (±1.4)
60.1 (±3 ) 6.3 (±0.06) 0.5 (±0.02)
^cat K']
1.61 (±0.02) 2.58(±0.04) 1.67 (±0.02) 1.70(±0.01)
0.69 1.11 0.71 0.76
DHFA NADH NADPH
[MM]
[MM]
[MM]
_ -
_
87.5
87.4
-
114 129
μ,πιοί nucleotide oxidised x min 1 X (mg protein) '.
-
1069
saturating concentrations of the second substrate and vice-versa. The parameters (Km, Vmax and kcat) were calculated using a computer program'141 and are listed in Table 3. Non-denaturing PAGE and enzyme activity staining Non-denaturing PAGE was carried out on 7.5% polyacrylamide slab gels at 4°C. The resolving gel composition was: acrylamide-bisacrylamide (30:0.8; 5 ml), 1.5MTris/HCl, pH 8.8buffer (3.75 m/), water (11.25 m/), ammonium persulphate (10%, 0.1 ml) andTemed (6 μΐ, Sigma). The stacking gel contained acrylamide-bisacrylamide (30:0.8; 1.25 ml), IM Tris/HCl, pH 6.7 (1.25 ml), water (6.25 ml), riboflavin (0.04%, 1.25 ml) and Temed (6 μΓ). The sample made 20% in glycerol was stacked at 60 Vfor l h and resolved at 120V for 3h. The gel staining procedure was essentially as described'15J with the modification that the mixture containing the substrates was made 0.3% inAgarose (Bio-Rad). This allowed the different enzyme activity stains to be carried out on one slab gel by using spacers to separate the different activities that were being investigated. The reagents for the DHPR activity stain were made in a gel as follows: agarose (0.03 g) was melted in 125rnMTris/HCl buffer pH 7.5 (7.5 m/) and mixed with NADH, quinonoid 6-methyl7,8(6//)-dihydropterin (prepared by mixing 6-methyl-5,6,7,8-tetrahydropterin with a 10% excess of 2,6-dichlorophenolindophenol (free acid), adjusting the pH to 7.0 and extracting the excess of dye with diethyl ether'16'), and MTTtetrazolium from Sigma at final concentrations of ΙΟΟμ-Μ, ΙΟΟμ,Μ and 200μ,Μ, respectively, and the whole mixture diluted to a final volume of 10 ml which was immediately layered on the polyacrylamide gel. This procedure is superior to dipping the acrylamide gel into an aqueous solution of the reagents. The DHFR stain solution was as above except that NADPH (ΙΟΟμ,Μ) was the reducing equivalent and 7,8-dihydrofolate (50μΜ) was the pterin substrate instead of NADH and quinonoid 6-methyldihydropterin. The pteridine-independent activity stain solution contained all the above reactants used in the DHPR stain except for quinonoid 6-methyldihydropterin. The gels were overlaid with the activity stain reactants in the respective tracks separated by spacers for 3-5 min and photographed (see Fig. 1). Analytical ultracentrifuge analysis E. coli DHPR (1 m/, 0.5 mg) was dialysed exhaustively against buffer A (3 x 1 /). Analytical ultracentrifugation using a meniscus depletion-type experiment was kindly carried out be Dr. P. Jeffrey (Protein Chemistry Group, JCSMR, ANU). The centrifugation speed was 40000 rpm at a controlled temperature of 20 ± 0.1 °C for 8h. The partial specific volume was assumed to be 0.73 m//g and the density was 1.001 g/m/ giving a calculated molecular mass of 25700 Da. The DHPR activity of the solution was measured after the experiment and was unchanged. Effect ofN,N-dimethylpropargyl amine on the three enzyme activities o f E . coli DHPR All measurements were carried out on a Gary 219 double-beam spectrometer thermostated at 25 °C and water was deionized in a Milli-Q system. For the oxidoreductase activity a stock solution of IM Tris/HCl, pH 7.4 (1 m/), water (7.35 m/), enzyme (20 μΐ, 0.25 μ-g) and dimethylpropargyl amine (2.2 mg, 3.1mM), and a similar solution without the amine were incubated at 25 °C. The blank solution contained iMTris/HCl, pH 7.4 (2 ml) and water (15 mf). For the assays aliquots (850 μΐ) were withdrawn at time intervals, 3mM potassium ferricyanide (100 μΐ, 0.3mM final concentration) was added and the reaction was started by adding 2mM NADH (50 μΐ, ΙΟΟμΜfinalconcentration), the blank cuvettes were treated similarly. The rate of change of absorbance at 340 nm was measured with the recorder set at 0 to 0.05 absorbance units. The initial rates of the solutions containing dimethylpropargyl amine decreased by 37% after 3.5h and
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S. Vasudevan, B. Paal und W.L.F. Armarego DHFR
1
DHPR
OR
2 3
4
5
6
7
8
9
10 11 12
O 50
25 18
Θ 1
2
3
4
5
6
7
8
9
10
11 12
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ductase activity of both solutions decreased slowly but at about equal rates. The rates had decreased to 40% in 2h, and 10% after 18h, of the original rates. For the dihydrofolate reductase activity a stock solution of iMTris/ HC1, pH 7.4 (2 m/), water (14.2 ml} and E. coli enzyme (800 μ/) was prepared, divided into two equal portions, one of which was added to dimethylpropargyl amine (4.5 mg, 6.4mM) and the second was the control, and were incubated at 25°C. The blank consisted of IM Tris/HCl, pH 7.4 (2 m/) and water (15 mO- For the assays, aliquots (850 μ/) were withdrawn at time intervals and NADPH (100 μ/, 45 μΜ final concentration) was added. The reactions were started by adding dihydrofolic acid (50 μ/, 118μΜ final concentration). The blank cuvettes were treated similarly. The rates of change of absorbance at 340 nm were measured as above. The initial rates of the DHFR reaction in the presence and absence of the dimethylpropargyl amine decreased very slowly with time. After 70h the activity had decreased by only 25% of the original value. However, on heating the solutions at 100°C for 10 min the DHFR activity in both cases dropped to 3% of the original value. Oxidoreductase activity The pterin-independent oxidoreductase activity (see above and ref.[5]) was recorded on a Gary 219 double beam spectrometer at 340 nm. Potassium ferricyanide behaved as a normal substrate and because the extinction coefficients of ferricyanide and ferrocyanide at 340 nm are very similar this could be used as the analytical wavelength. The Km, Vmax and &ΜΓ values for potassium ferricyanide were found to be 30.5 (±2.8) μΜ and 8.77 (±0.2) μηιοί NADH oxidised x min"1 x (mg protein)"1 and 3.7s~J, respectively. Amino acid analysis
Fig. 1. Activity staining of DHFR, pterin-independent oxidoreductase (OR) and DHPR activies. A) The enzyme activities were stained as described in the Materials and Methods section. Lanes 1, 5 and 9 contain purified human DHPR (5 μg each); lanes 2, 6 and 10 and lanes 3,7 and 11 contain E. coli DHPR (0.75 and 3.0 /x,g, respectively); lanes 4, 8 and 12 contain E. coli DHFR (3 μ-g each). The light spots in the centre of the DHFR spots (lanes 4 and 9) are possibly due to the high reductase activity which may have reduced the coloured formazan further to the less coloured hydrazidine and amidrazone derivatives of MTT. B)The gel from A stained for protein with Coomassie Blue.
Samples of E. coli DHPR (25 μg) were exhaustively dialysed against buffer A (2 x 500 ml) and water (2 x 500 ml) before hydrolysing for 24,48 and 72 h in OM HC1 and were analysed on a Beckman System 6300. The composition is in Table 4.
Table4. Comparison of E. coli DHPR amino-acid composition with rat and human liver DHPR.
Lys His Arg Cys Asx Thr Ser Glx Pro Gly Ala Val Met He Leu Tyr Phe Trp
by 94% of the original value after 18 h. The activity of the solution containing the enzyme but free from the amine was unaltered after 7 h. For the dihydropteridine reductase activity a stock solution of IM Tris/HCl, pH 7.4 (2 m/), water (12.4 ml) and enzyme (100μ/) was divided into two equal portions, one of which was added to dimethylpropargyl amine (3.5mM final concentration) and the other was for the control without amine, and incubated at 25°C. The blank solution contained IM Tris/HCl, pH 7.4 (2 mO and water (6.5 m/). For the assays, peroxidase (100 μ/, 20 μ-g), hydrogen peroxide (50 μ/, 11 μπιοί) and 6-methyl-5,6,7,8-tetrahydro-(3//)pterin hydrochloride (100 μ/ in 5mM HC1, ΙΟΟμΜ final concentration) were added to aliquots (750 μ/) which were withdrawn at time intervals. The reactions were initiated by addition of 2mM NADH (50 μ/, final concentration ΙΟΟμΜ). The blank cuvettes were treated similarly and the initial rates were determined as above at 340 nm. After incubation for 5 min the initial rate of the solution containing dime thylpropargyl amine was 60% of the initial rate of the control solution without the amine, but thereafter the dihydropteridine re-
Residues/subunit
Residue E. coli
Human"
Rata
17 5 8 3C 24 11 11 27 8 25 29 19 3(6) 13 19 3 7 N.D.
15 4 8 4 19 17 20 19 8 25 29 18 7 9 21 3 7 7
14 4 9 4 16 17 19 21 9 25 33 18 7 9 21 3 7 7
N.D. Not determined. Deduced from rat liver cDNAnucleotide sequence. b Deduced from a human liver cDNAnucleotide sequence. c Determined as cysteic acid. a
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Vol. 373 (1992)
E. coli Dihydropteridine Reductase with Dihydrofolate Reductase activity
Results and Discussion The purification of E. coli DHPR described previously involved three column purification steps including two FPLC column (Mono Q and Mono P) separations. The observation that DTT destabilised the reductase and the report that flavoenzymes are more stable in Hepes buffer[17] prompted us to re-evaluate our purification scheme (see Material and Methods). The purification profile inTable 1 shows an overall activity yield of 40%. Increased stability of the reductase (flavoprotein) in Hepes buffer gave better recovery during ammonium sulphate fractionation and the DEAE-Fractogel with bead properties that prevent shrinkage gave good yields of active fractions. Preparative isoelectric focusing was used in preference to FPLC Mono P chromatofocusing because of the large sample weight and volume that could be applied. Also at the end of the focusing the reductase appeared as a sharp bright yellow band that was easily isolated. The specific activity of E. coli DHPR purified by this method was of the same order as we reported earlier. This preparation has been stored in Hepes buffer pH 7.4 with 20μΜ NADH at -20°C for more than one year without loss of activity. Stability of DHPR Most dihydropteridine reductases are stable for several years in the presence of 2mM DTT and 20μΜ NADH in 50mMTris/HCl pH 7.4 at -20°C[13] and this has been our experience with human brain enzyme. In the early studies of the preparation of E. coli DHPR we found that the reductase lost half of its activity upon storage at -70°C. When NADH (20μΜ final concentration) was added to the pooled fractions from the DEAE anion exchange purification step, the light yellow colour had disappeared instantly, but reappeared upon standing in air without loss of enzyme activity. However when DTT (2mM final concentration) was added to the pooled fracions, 30% of enzyme activity was lost. A more detailed investigation showed that unlike with human reductase, 2mM DTT (final concentration) inactivates the enzyme bothat -20°Cand25°Cwith30% and77% lossof activity, respectively, after 24 h. NADH (20μΜ) protects the enzyme from DTTinactivation at both temperatures. The stability of the purified enzyme under various conditions is summarised in Table 2. The mechanism of DTT inactivation is not clear but it is known that DTTcan reduce FAD and can act as a substrate for some flavoproteins[17]. In view of the above results the general practice of adding DTT to stabilize flavin-containing proteins needs to be reconsidered.
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Presence of dihydrofolate reductase activity Dihydropteridine reductase and dihydrofolate reductase reduce two different tautomers of dihydropterin (1 and 3, respectively, in Scheme 1). Generally DHPRs will not use 7,8-dihydrofolate or 7,8-dihydro(3//)-pterins as substrates and DHFRs do not reduce quinonoid 7,8-dihydro-(6//)-pterins. We have confirmed that no reaction occurred when human brain DHPR was added to 7,8-dihydrofolate (118μΜ) in the presence of NADH (ΙΟΟμ,Μ) at pH 7.4; and E. coli DHFR was inactive in the presence of quinonoid 6methyl-7,8-dihydro-(6//)-pterin (ΙΟΟμ,Μ) and NADPH (54μΜ). There was also no detectable DHPR activity in the R-plasmid R67 encoded DHFR[18] which bears no sequence homology to other DHFRs. The present E. coli DHPR was found to have dihydrofolate reductase activity which was confirmed by determining the kinetic parameters with NADH and NADPH separately as nucleotide cofactors (see Table 3). This reductase, like known dihydrofolate reductases^, but unlike dihydropteridine reductases^, has a Km value for NADPH that is less, by one order of magnitude, than that for NADH. Comparison of DHFR-specific activities in cell-free extracts of the DHFR~ mutant D3-157 (2.54 mU/mg protein using NADPH and 7.19 mU/mg using NADH) with those from the parent strain H712 (wildtype; 28.8 mU/mg protein using NADPH and 6.27 mU/mg using NADH) and a DHFR over-producing strain JFM 223 (729 mU/mg protein using NADPH and 24.3 mU/mg usning NADH) were made. Despite the relatively high level of DHFR activity in the DHFR-deficient strain, which is most probably due to the DHPR, this strain was unable to grow in minimal media without folate-dependent end-products, e.g. thymidine^10'2^. This raises the question of the in vivo capacity of the E. coli DHPR to carry out the function of DHFR. To address this question we attempted to clone the E. coli DHPR gene[22] in order to express the reductase in high levels in D3-157 (DHFR~) to see if the strain would now grow in minimal media. Using an expression cloning vector strategy we isolated a gene that codes for a hemoglobinlike protein with DHPR activity which is different from the present 25.7-kDa protein. Enzyme activity stain The basis of the enzyme activity staining method is that tetrahydro- pterins and folate which are the products of DHPR- and DHFR-catalysed reactions can reduce the soluble tetrazolium MTTdye to yield the insoluble blue formazan^23'24!. The rate of formation of the blue colour is dependent on the reduction potential of the products of the enzymic reactions. The acBrought to you by | Purdue University Libraries Authenticated Download Date | 5/23/15 5:31 AM
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S. Vasudevan, B. Paal und W.L.F. Armarego
tivity staining of the native gel clearly shows that human DHPR does not possess DHFR activity whereas E. coli DHFR and E. coli DHPR both stain positively because tetrahydrofolate reduces MTT rapidly. Human DHPR and E. coli DHPR stain positively for DHPR also because tetrahydropterins formed reduced MTT. It became evident that E. coli DHPR can utilise tetrazolium MTT as an artificial electron acceptor in the absence of folates and pterins and produces a blue colour but more slowly than in the above case. This allowed the pterin-independent oxidoreductase activity to be stained blue on the gels. Human DHPR and E. coli DHFR, not being flavoproteins do not reduce MTT in the absence of pterins and folate, and are not stained blue with the oxidoreductase activity stain (Fig. 1). Molecular mass The activity stain gel (Fig. 1) also shows clearly the molecular mass of the active E. coli DHPR is greater than 18.5 kD (molecular mass DHFR) and much less than 50 kDa (molecular mass of active dimeric human DHPR). The activity staining comparisons suggest that the molecular mass of native E. coli DHPR is the same as that of its subunit (25.7 kDa). Gel filtration studies and comparing the elution time of E. coli DHPR with standard proteins in 50mM Tris/HCl pH 7.4 containing 2mM DTTand 20mM NADH indicated that the protein existed as a dimer with a molecular mass of 54 ±2 kDa^. This is conflicting with the data from the activity staining and gel filtration experiments and prompted the determination of the molecular mass by analytical centrifugation. The analytical ultracentrifugation analysis using the meniscus depletion experiment^255 in 50mM Hepes pH 7.4 at 40 k rpm. (20 °C for 8h) showed that the bulk of the protein was concentrated in a region where its molecular mass (calculated) was 25.7 kDa assuming the density of solution was 1.001 g/m/ and the partial specific volume was 0.73 m//g. It is likely that the gel filtration value may have been over-estimated because of retardation brought about by reactivity with DTT. The trifunctionality of E. coli DHPR The purified enzyme has been shown to possess three separate activities, viz pterin-independent NADH oxidoreductase, dihydropteridine reductase and dihydrofolate reductase activities. By using saturating concentrations of substrates i.e. concentrations at which the initial rates are maximal, and the same amounts of enzyme and NADH (ΙΟΟμΜ), the ratio of the three activities pterin-independent oxidoreductase, DHPR and DHFR are 4.3:1.0:0.05, respectively. The amino-acid composition was determined
Vol. 373 (1992)
and when compared with that of the rat and the human DHPR (seeTable 4) there was superficial similarity in composition. The molecular mass (25.7 kDa) was closer to the subunit molecular mass of human DHPR (active as dimer of subunit molecular mass 50 kDa[26)) than the molecular mass of E. coli DHFR (18.5 kDa), but like the latter it is active as a monomer. Attempts to isolate the gene coding for the E. coli reductase by expression cloning using a cosmid library resulted in the discovery of a novel 44 kDa flavo-haemoglobin with dihydropteridine reductase activity[22]. Much is known about the mechanism of reduction of the non-flavin containing DHFR[19] and DHPR[13 271 but the presence of FAD in a protein possessing both these activities led us to examine the effect of the well known irreversible flavin inhibitor, A^TV-dimethylpropargyl amine[28~30] on all three activities. The amine did not inhibit the activities equally (see Materials and Methods). The pterin-independent oxidoreductase activity was weakly inhibited compared with other flavoproteins (e.g. ref.[29]), the DHPR activity immediately decreased by 40% and thereafter was very slowly inhibited, and the DHFR activity was not affected.These data imply that the flavin may not be involved in all three reactions. The presence of all three activities in the same protein prompts the speculation that mammalian dihydrofolate reductases and dihydropteridine reductases may have evolved from the same oxidoreductase flavoprotein. References 1 2 3 4 5 6 7 8 9
Shiman, R. (1985) in Folates and Pterins vol. 2 (Blakley, R.L. & Benkovic, S.J., eds.), pp. 179-249, J. Wiley & Sons, New York. Kaufman, S. & Kaufman, E.E. (1985) in Folates and Pterins vol. 2 (Blakley, R.L. & Benkovic, S.J., eds.), pp. 251-352, J.Wiley & Sons, New York. K hn, D.M. & Lovenberg, W. (1985) in Folates and Pterins vol. 2 (Blakley, R.L. & Benkovic, S.J., eds.), pp. 353-399, J. Wiley & Sons, New York. Armarego, W.L.F. & Kosar-Hashemi, Β. (1989) in Chemistry and Biology of Pteridines (Curtius, H.-C., Gisla, S. & Blau, N., eds.), pp. 620-623,W. de Gruyter, Berlin. Vasudevan, S.G., Shaw, D.C. & Armarego, W.L.F. (1988) Biochem. J. 255, 581-588. Dahl, H.-H.M., Hutchinson,W, McAdam, W.,Wake, S., Morgan, F.J. & Cotton, R.G.H. (1987) Nucl. Acids Res. 15,25-28. Lockyer, J., Cook, R.G., Milstein, S., Kaufmann, S., Woo, S.L.C. & Ledley, F.D. (1987) Proc. Natl. Acad. Sei. USA 84, 3329-3333. Shahbaz, M., Hoch, J.A., Trach, K.A., Hural, J.A., Webber, S. & Whiteley, J.M. (1987) J. Biol. Chem. 262, 16412-16416. Armarego,W.L.F. &Vasudevan, S.G. (1989) in Chemistry and Biology of Pteridines (Curtius, H.-Ch., Gisla, S. & Blau, N., eds.) pp. 616-619.
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E. coli Dihydropteridine Reductase with Dihydrofolate Reductase activity
10 Singer, S., Ferone, R., Walton, L. & Elwell, L. (1985) /. Bacterial 164, 470-472. 11 Smith, D.R., Rood, J.I., Bird, P.I., Sneddon, M.K., Calvo, J.M. & Morrison, J.F. (1982) /. Biol. Chem. 257, 9043-9048. 12 Armarego, W.L.F. & Schou, H. (1978) Aust. J. Chem. 31, 1081-1094. 13 Armarego, W.L.F., Randies, D. & Waring, P. (1984) Med. Res. Rev. 4, 267-321. 14 Cornish-Bowden, A. & Endrenyi, L. (1981) Biochem. J. 193,1005-1006. 15 Cotton, R.G.H. & Jennings, I. (1978) Eur. J. Biochem. 83, 319-324. 16 Kaufman, S. (1961) J. Biol. Chem. 236,804-810. 17 Desa, R.J. (1976) in Flavins and Flavoproteins (Singer, T.P., ed.) pp. 720-725, Elsevier, Amsterdam. 18 Matthews, D.A., Smith, S.L., Baccanari, D.P., Burchall, J.J., Oatley, S.J. & Kraut, J. (1986) Biochemistry 25, 4194-4204. 19 Blakley, R.L. (1984) in Folates and Pterins vol. 1 (Blakley, R.L. & Benkovic, S.J., eds.) pp. 191-253.
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20 Armarego,W.L.F. (1989) Pteridines 1,179-188. 21 Vasudevan, S.G. (1989) Ph.D.Thesis, Australian National University. 22 Vasudevan, S.G., Armarego, W.L.F., Shaw, D.C., Lilley, P.E., Dixon, N.E. & Poole, R.K. (1991) Mol Gen. Genet. 266,49-58. 23 Hooper,WD. (1969) Pure andAppl Chem. 19,221-241. 24 Gunlak, E.G., Neal, G.E. & Williams, D.C. (1966) Biochem. J. 101,29P-30P. 25 Yphantis, D.D. (1964) Biochemistry 3,295-316. 26 Firgaira, F, Cotton, R.G.H. & Danks, D. (1981) Biochem. J. 197, 31-43. 27 Randies, D. (1986) Eur. J. Biochem. 155, 301-304. 28 Gisla, S., Wenz, A. &Thorpe, C. (1980) in Enzyme Inhibitors (Brodbeck, U., ed.) pp. 43-60, Verlag Chemie, Basel. 29 Maycock, A.L., Abeles, R.H., Salach, J.I. & Singer, T.P. (1976) Biochemistry 15,114-125. 30 Abeles, R.H. (1978) in Enzyme-Activated Irreversible Inhibitors (Seiler, N., Jung, M.J. & Koch-Weser, J., eds.) pp. 1-12, Elsevier/North Holland, Amsterdam.
S.G. Vasudevan3, B. Paal and W.L.F. Armarego*b, Research School of Chemistry3 and Protein Biochemistry Group15, Division of Biochemistry and Molecular Biology, The John Curtin School of Medical Research, The Australian National University, G.P.O. Box 4, Canberra City, ACT 2601, Australia. * To whom correspondence should be addressed.
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Dihydropteridine Reductase from Escherichia coli Exhibits Dihydrofolate Reductase Activity Subhash G. VASUDEVAN*, Bela ?AALb and Wilfred L.F. ARMAREGO b Research School of Chemistry3 and Protein Biochemistry Group b , Division of Biochemistry and Molecular Biology, The John Curtin School of Medical Research, The Australian National University, Canberra City, Australia (Received 10 August 1992)*
Summary: E. coli Dihydropteridine reductase, known to have a pterin-independent oxidoreductase activity with potassium ferricyanide as electron donor, has now been shown to possess also dihydrofolate reductase activity. The kinetic parameters for dihydrofolate reductase activity have been determined. The ratio of the three activities, dihydropteridine reductase, dihydrofolate reductase and pterin-independent oxidoreductase activity is 1.0, 0.05 and 4.3, respectively. The enzyme, a flavoprotein which is unstable in the presence of dithiothreitol, was shown to be a
monomer with a molecular mass of 25.7 kDa. The apparent lack of discrimination between hydride transfer from the pyridine nucleotide to N5 of the pterin in the dihydropteridine reductase reaction and C6 of folate in the dihydrofolate reaction suggested that the FAD prosthetic group may be involved in the hydride transfers. The flavoprotein inhibitor N,N- dimethylpropargylamine inhibited the dihydropteridine reductase and oxidoreductase reactions differently and did not affect the dihydrofolate reductase activity however.
Key terms: Dihydropteridine reductase, dihydrofolate reductase, flavoprotein, hydride transfer, /V,yV-dimethylpropargylamine, E. coli.
Dihydropteridine reductase (EC. 1.6.99.7, DHPR) catalyses the reduction of "quinonoid" dihydrobiopterin. The latter is the natural cofactor for phenylalanine (EC 1.14.16.1)[1], tyrosine (EC 1.14.16.2)[2] and tryptophan (EC 1.14.16.4)[3] hydroxylases, and also for other enzymic reactions that require further
characterization e.g. glyceryl etherase (EC 1.14.16.5)^. A reductase from Escherichia coli with dihydropteridine reductase activity has been isolated to electrophoretic homogeneity. Unlike other dihydropteridine reductases that have been studied E. coli reductase possesses an FAD prosthetic
Enzymes : Dihydrofolate reductase, 5,6,7,8-tetrahydrofolate:NADP® oxidoreductase (EC 1.5.1.3); Dihydropteridine reductase, NAD(P)H:6,7-dihydropteridine oxidoreductase (EC 1.6.99.7); Glyceryl-ether monooxygenase, 1-alkyl-sn-glycerol, tetrahydropteridine:oxygen oxidoreductase (EC 1.14.16.5); Phenylalanine 4-monooxygenase, L-phenylalanine, tetrahydrobiopterin:oxygen oxidoreductase (4-hydroxylating) (EC 1.14.16.1) (in this paper named phenylalanine hydroxylase); Tryptophan 5-monooxygenase, L-tryptophan, tetrahydropteridine:oxygen oxidoreductase (5-hydroxylating) (EC 1.14.16.4) (in this paper named tryptophan hydroxylase); Tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating) (EC 1.14.16.2) (in this paper named tyrosine hydroxylase). Abbreviations: DHFR, dihydrofolate reductase; DHPR, dihydropteridine reductase; DTT, dithiothreitol; FPLC, fast-protein liquid chromatography; Hepes, 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide; Temed, Ν, Ν, Ν', Ν' -tetramethylethylenediamine. * Received by PTERIDINES 24 April 1992
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S. Vasudevan, B. Paal und W.L.F. Armarego
The superficial similarities between DHPR and dihydrofolate reductase (EC 1.5.1.3, DHFR) catalysed reactions (see Scheme 1) suggest that the two enzymes may have evolved from a common precursor and hence may share some structural similarities. Human liver cDNA[6J1 and a rat-liver cDNA[8] coding for DHPR have been cloned and sequenced. The amino-acid sequence deduced for the human-liver DHPR shows very little homology with that of human DHFR except for a short region around Cys104 for the former and Gly20 in the latter which is known to be important for pyridine nucleotide binding^. The extensive sequence identity between rat and human DHPR (only 10 conservative changes) implies that the structure of this enzyme is crucial for its function. In this paper we describe a purification procedure that gives a higher yield of the reductase from E. coli and present detailed evidence that the enzyme has DHFR activity. This is the first known enzyme to possess these two activities in the same protein. Part of this work was communicated at an international pteridine conference in Ziirich[9]. In contrast to our previous report the enzyme is functional as a protein of 25700 Da molecular mass and does not exist as a dimer. NADH,
NAD* H
H
NADP*
SCHEME 1
Materials and Methods E. coli K-12 derivative strains H712 and D3-157[10], and JFM 228'11' were used in this study. Purified E. coli DHFR and R-plasmid encoded DHFR were gifts from Dr. John F. Morrison (ANU). The 5,6,7,8-tetrahydropterins used in the assays were prepared by known procedures'12'. Chemicals of analytical or higher grades were used wherever possible. Growth of cells and preparation of cell-free extracts The media used and the method of growth for large scale enzyme preparations were as described'5'. Ampicillin (Beecham Research
Vol. 373 (1992)
Laboratories, Australia) was used at 50 μξ/ml for growth of JFM 228. The cells obtained after centrifugation were washed with 50mM Hepes/KOH pH 7.4 containing ImM EDTAand 200mM KCl and the cell-free extract was obtained as described previously'5'. Enzyme purification All manipulations were carried out at 4°C unless otherwise stated. The cell-free exract was fractionated with solid ammonium sulphate (84% of the DHPR activity was obtained in the 35-45% ammonium sulphate saturation) followed by centrifugation (150000xg). The precipitate was resuspended in 50mM Hepes/ KOH pH 7.4 (buffer A) and dialysed over-night against buffer A (3 x 2 /). The dialysate was applied onto a Fractogel TSK DEAE650 (M) column (2.0 x 15 cm, from Merck) which was equilibrated with buffer A. The column was washed with 10 column volumes of buffer A followed by elution with a linear gradient formed with 250 ml of buffer A and 250 ml of buffer A containing 200mM NaCl. The enzyme activity eluted at about lOOmM NaCl. The peak active fractions were pooled and the total protein was precipitated with ammonium sulphate (80% saturation) and pelleted by centrifugation. The pellet was re-suspended in buffer A and dialysed against it (3 x 500 ml). For long-term storage 20/iM NADH was added at this stage. When used immediately for the next purification step the pellet was dissolved in water (5 ml). The enzyme solution (2.5 m/) was loaded immediately on to two PD-10 columns (Sephadex G-25 pre-packed columns, Pharmacia) that have been pre-equilibrated with water. The combined eluates from two purifications (2x3.5 m/) of enzyme were made to 4% in ampholines (0.15 ml of Biolyte 3/10,40% w/v and 0.35 ml of Biolyte 5/7,40%; w/v from LKB) and applied to a preparative isoelectric focusing slab set up essentially as described by the manufacturer (Bio-Rad, Bio-Phoresis Instruction Manual). Bio-lyte electrofocusing gel (Bio-Rad 150 m/) made 4% in ampholytes (6 m/Biolyte 3/10,40% w/v and 9 m/Biolyte 5/7, 40% w/v) was set into a gel tray (20 x 11 x 0.8 cm) by wicking off the excess liquid from both ends of the tray. The sample was applied in two additions in the middle of the tray and wicking at the ends. The anode (0.33M H3PO4) and cathode (lM NaOH) were applied on thin-filter strips and placed at each end of the tray. Electrofocusing was carried out in Biophoresis cells (Bio-Rad) at constant voltage of 250 V for 2 h followed by 18 h at 700V. The yellow colour of the prosthetic group enabled easy visualization of the reductase which was closer to the anodic end. The yellow band was removed with a spatula, extracted with buffer A (3 x 5 ml) containing 200mM NaCl and centrifuged at 50000 x g min. The active functions were pooled and dialysed against buffer A (2 x 500 ml). The enzyme was stored in 1 ml aliquots with 20μΜ NADH at -20°C. The purification steps are summarised in Table 1. One activity unit (U) corresponds to μηιοί NADH oxidised x min""1 x (ml of enzyme)"1. Enzyme stability The purified protein (25 /xg, 50 μΐ) was incubated with various additives at 25 °C and -20°C (Table 2). The samples were thawed and 10 μΐ of sample was removed and assayed for DHPR activity'13' every 24 h. Dihydrofolate reductase assay The assay mixture contained: iMTris/HCl pH 7.4 (100 μ[), NADPH (ΙΟΟμΜ) and 7,8-dihydrofolate (ΙΟΟμ-Μ) in a total volume of 1 ml per cuvette at 25 °C. The reaction was initiated by addition of the enzyme (20 μΙ; 0.25 //.g of purified protein) into one cuvette. The initial rates were obtained from the rate of decrease of absorbance at 340 nm (ε for NAD(P)H is 620θΜ-1 cm"1). Note that the rate of decrease in absorbance is two times the real rate because 7,8-dihydrofolate absorbs at 340 nm with the same ε value. Thus a correction factor of 0.5 is used to obtain the rate of consumption of NADPH for the DHFR reaction. The kinetic parameters were obtained from the initial rates at various concentrations of one substrate and
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Vol. 373 (1992)
E. coli Dihydropteridine Reductase with Dihydrofolate Reductase activity
Table 1. Purification of DHPR from E. coli. Purification steps3 Volume
[m/] Cell-free extract Ammonium sulphate fraction 6 DEAEanion exchange0 Preparative isoelectric focusingd a
Total Total Spec. activity protein activity e [U] [U/mg]
Yield
[%]
100
303
5830
0.05
40
256
1350
0.19
84.5
26
210
142
1.9
63
9
120
27
4.5
100
40
Hepes KOH (pH 7.4) was used throughout the purification. A 35-45% ammonium sulphate fraction was used. DEAE-Fractogel. Using the Bio-Phoresis system (Βίο-Rad) with a pH gradient of4.5-7.5. For definition of U see Material and Methods.
b c d e
Table 2. Stability of DHPR from E. coli. Conditions3
ra DHPR without addition DHPR + DTT (2mM) DHPR + DTT (2mM) DHPR + NADH DHPR + NADH (20μΜ) DHPR + DTT(2mM) + NADH (20μΜ) DHPR -f DTT(2mM) + ΝΑΟΗ(20μΜ) a b c
DHPR activity [U]b after
Temp.
0.0 hc
24 h
48 h
72h
7.8
6.9
7.3
6.8
-20
5.3
3.7
2.8
2.3
25
5.3
1.2
0.3
0.0
-20
6.9
7.1
7.1
7.1
25
6.5
7.2
6.8
6.5
-20
5.8
5.7
5.4
4.7
25
5.8
5.3
4.6
3.8
20
In Hepes KOH (pH 7.4). For definition of U see Material and Methods. The activity at time = 0 h was carried out to correct for reduction of activity due to volume changes.
Table3. Dihydrofolate reductase activity of E. coli DHPR O.lM Tris/HCl,pH7.4at25°. Substrate
Km
Vmax3
[MM]
DHFA DHFA NADH NADPH a
27.3 (±1.4)
60.1 (±3 ) 6.3 (±0.06) 0.5 (±0.02)
^cat K']
1.61 (±0.02) 2.58(±0.04) 1.67 (±0.02) 1.70(±0.01)
0.69 1.11 0.71 0.76
DHFA NADH NADPH
[MM]
[MM]
[MM]
_ -
_
87.5
87.4
-
114 129
μ,πιοί nucleotide oxidised x min 1 X (mg protein) '.
-
1069
saturating concentrations of the second substrate and vice-versa. The parameters (Km, Vmax and kcat) were calculated using a computer program'141 and are listed in Table 3. Non-denaturing PAGE and enzyme activity staining Non-denaturing PAGE was carried out on 7.5% polyacrylamide slab gels at 4°C. The resolving gel composition was: acrylamide-bisacrylamide (30:0.8; 5 ml), 1.5MTris/HCl, pH 8.8buffer (3.75 m/), water (11.25 m/), ammonium persulphate (10%, 0.1 ml) andTemed (6 μΐ, Sigma). The stacking gel contained acrylamide-bisacrylamide (30:0.8; 1.25 ml), IM Tris/HCl, pH 6.7 (1.25 ml), water (6.25 ml), riboflavin (0.04%, 1.25 ml) and Temed (6 μΓ). The sample made 20% in glycerol was stacked at 60 Vfor l h and resolved at 120V for 3h. The gel staining procedure was essentially as described'15J with the modification that the mixture containing the substrates was made 0.3% inAgarose (Bio-Rad). This allowed the different enzyme activity stains to be carried out on one slab gel by using spacers to separate the different activities that were being investigated. The reagents for the DHPR activity stain were made in a gel as follows: agarose (0.03 g) was melted in 125rnMTris/HCl buffer pH 7.5 (7.5 m/) and mixed with NADH, quinonoid 6-methyl7,8(6//)-dihydropterin (prepared by mixing 6-methyl-5,6,7,8-tetrahydropterin with a 10% excess of 2,6-dichlorophenolindophenol (free acid), adjusting the pH to 7.0 and extracting the excess of dye with diethyl ether'16'), and MTTtetrazolium from Sigma at final concentrations of ΙΟΟμ-Μ, ΙΟΟμ,Μ and 200μ,Μ, respectively, and the whole mixture diluted to a final volume of 10 ml which was immediately layered on the polyacrylamide gel. This procedure is superior to dipping the acrylamide gel into an aqueous solution of the reagents. The DHFR stain solution was as above except that NADPH (ΙΟΟμ,Μ) was the reducing equivalent and 7,8-dihydrofolate (50μΜ) was the pterin substrate instead of NADH and quinonoid 6-methyldihydropterin. The pteridine-independent activity stain solution contained all the above reactants used in the DHPR stain except for quinonoid 6-methyldihydropterin. The gels were overlaid with the activity stain reactants in the respective tracks separated by spacers for 3-5 min and photographed (see Fig. 1). Analytical ultracentrifuge analysis E. coli DHPR (1 m/, 0.5 mg) was dialysed exhaustively against buffer A (3 x 1 /). Analytical ultracentrifugation using a meniscus depletion-type experiment was kindly carried out be Dr. P. Jeffrey (Protein Chemistry Group, JCSMR, ANU). The centrifugation speed was 40000 rpm at a controlled temperature of 20 ± 0.1 °C for 8h. The partial specific volume was assumed to be 0.73 m//g and the density was 1.001 g/m/ giving a calculated molecular mass of 25700 Da. The DHPR activity of the solution was measured after the experiment and was unchanged. Effect ofN,N-dimethylpropargyl amine on the three enzyme activities o f E . coli DHPR All measurements were carried out on a Gary 219 double-beam spectrometer thermostated at 25 °C and water was deionized in a Milli-Q system. For the oxidoreductase activity a stock solution of IM Tris/HCl, pH 7.4 (1 m/), water (7.35 m/), enzyme (20 μΐ, 0.25 μ-g) and dimethylpropargyl amine (2.2 mg, 3.1mM), and a similar solution without the amine were incubated at 25 °C. The blank solution contained iMTris/HCl, pH 7.4 (2 ml) and water (15 mf). For the assays aliquots (850 μΐ) were withdrawn at time intervals, 3mM potassium ferricyanide (100 μΐ, 0.3mM final concentration) was added and the reaction was started by adding 2mM NADH (50 μΐ, ΙΟΟμΜfinalconcentration), the blank cuvettes were treated similarly. The rate of change of absorbance at 340 nm was measured with the recorder set at 0 to 0.05 absorbance units. The initial rates of the solutions containing dimethylpropargyl amine decreased by 37% after 3.5h and
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1070
S. Vasudevan, B. Paal und W.L.F. Armarego DHFR
1
DHPR
OR
2 3
4
5
6
7
8
9
10 11 12
O 50
25 18
Θ 1
2
3
4
5
6
7
8
9
10
11 12
Vol. 373 (1992)
ductase activity of both solutions decreased slowly but at about equal rates. The rates had decreased to 40% in 2h, and 10% after 18h, of the original rates. For the dihydrofolate reductase activity a stock solution of iMTris/ HC1, pH 7.4 (2 m/), water (14.2 ml} and E. coli enzyme (800 μ/) was prepared, divided into two equal portions, one of which was added to dimethylpropargyl amine (4.5 mg, 6.4mM) and the second was the control, and were incubated at 25°C. The blank consisted of IM Tris/HCl, pH 7.4 (2 m/) and water (15 mO- For the assays, aliquots (850 μ/) were withdrawn at time intervals and NADPH (100 μ/, 45 μΜ final concentration) was added. The reactions were started by adding dihydrofolic acid (50 μ/, 118μΜ final concentration). The blank cuvettes were treated similarly. The rates of change of absorbance at 340 nm were measured as above. The initial rates of the DHFR reaction in the presence and absence of the dimethylpropargyl amine decreased very slowly with time. After 70h the activity had decreased by only 25% of the original value. However, on heating the solutions at 100°C for 10 min the DHFR activity in both cases dropped to 3% of the original value. Oxidoreductase activity The pterin-independent oxidoreductase activity (see above and ref.[5]) was recorded on a Gary 219 double beam spectrometer at 340 nm. Potassium ferricyanide behaved as a normal substrate and because the extinction coefficients of ferricyanide and ferrocyanide at 340 nm are very similar this could be used as the analytical wavelength. The Km, Vmax and &ΜΓ values for potassium ferricyanide were found to be 30.5 (±2.8) μΜ and 8.77 (±0.2) μηιοί NADH oxidised x min"1 x (mg protein)"1 and 3.7s~J, respectively. Amino acid analysis
Fig. 1. Activity staining of DHFR, pterin-independent oxidoreductase (OR) and DHPR activies. A) The enzyme activities were stained as described in the Materials and Methods section. Lanes 1, 5 and 9 contain purified human DHPR (5 μg each); lanes 2, 6 and 10 and lanes 3,7 and 11 contain E. coli DHPR (0.75 and 3.0 /x,g, respectively); lanes 4, 8 and 12 contain E. coli DHFR (3 μ-g each). The light spots in the centre of the DHFR spots (lanes 4 and 9) are possibly due to the high reductase activity which may have reduced the coloured formazan further to the less coloured hydrazidine and amidrazone derivatives of MTT. B)The gel from A stained for protein with Coomassie Blue.
Samples of E. coli DHPR (25 μg) were exhaustively dialysed against buffer A (2 x 500 ml) and water (2 x 500 ml) before hydrolysing for 24,48 and 72 h in OM HC1 and were analysed on a Beckman System 6300. The composition is in Table 4.
Table4. Comparison of E. coli DHPR amino-acid composition with rat and human liver DHPR.
Lys His Arg Cys Asx Thr Ser Glx Pro Gly Ala Val Met He Leu Tyr Phe Trp
by 94% of the original value after 18 h. The activity of the solution containing the enzyme but free from the amine was unaltered after 7 h. For the dihydropteridine reductase activity a stock solution of IM Tris/HCl, pH 7.4 (2 m/), water (12.4 ml) and enzyme (100μ/) was divided into two equal portions, one of which was added to dimethylpropargyl amine (3.5mM final concentration) and the other was for the control without amine, and incubated at 25°C. The blank solution contained IM Tris/HCl, pH 7.4 (2 mO and water (6.5 m/). For the assays, peroxidase (100 μ/, 20 μ-g), hydrogen peroxide (50 μ/, 11 μπιοί) and 6-methyl-5,6,7,8-tetrahydro-(3//)pterin hydrochloride (100 μ/ in 5mM HC1, ΙΟΟμΜ final concentration) were added to aliquots (750 μ/) which were withdrawn at time intervals. The reactions were initiated by addition of 2mM NADH (50 μ/, final concentration ΙΟΟμΜ). The blank cuvettes were treated similarly and the initial rates were determined as above at 340 nm. After incubation for 5 min the initial rate of the solution containing dime thylpropargyl amine was 60% of the initial rate of the control solution without the amine, but thereafter the dihydropteridine re-
Residues/subunit
Residue E. coli
Human"
Rata
17 5 8 3C 24 11 11 27 8 25 29 19 3(6) 13 19 3 7 N.D.
15 4 8 4 19 17 20 19 8 25 29 18 7 9 21 3 7 7
14 4 9 4 16 17 19 21 9 25 33 18 7 9 21 3 7 7
N.D. Not determined. Deduced from rat liver cDNAnucleotide sequence. b Deduced from a human liver cDNAnucleotide sequence. c Determined as cysteic acid. a
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Vol. 373 (1992)
E. coli Dihydropteridine Reductase with Dihydrofolate Reductase activity
Results and Discussion The purification of E. coli DHPR described previously involved three column purification steps including two FPLC column (Mono Q and Mono P) separations. The observation that DTT destabilised the reductase and the report that flavoenzymes are more stable in Hepes buffer[17] prompted us to re-evaluate our purification scheme (see Material and Methods). The purification profile inTable 1 shows an overall activity yield of 40%. Increased stability of the reductase (flavoprotein) in Hepes buffer gave better recovery during ammonium sulphate fractionation and the DEAE-Fractogel with bead properties that prevent shrinkage gave good yields of active fractions. Preparative isoelectric focusing was used in preference to FPLC Mono P chromatofocusing because of the large sample weight and volume that could be applied. Also at the end of the focusing the reductase appeared as a sharp bright yellow band that was easily isolated. The specific activity of E. coli DHPR purified by this method was of the same order as we reported earlier. This preparation has been stored in Hepes buffer pH 7.4 with 20μΜ NADH at -20°C for more than one year without loss of activity. Stability of DHPR Most dihydropteridine reductases are stable for several years in the presence of 2mM DTT and 20μΜ NADH in 50mMTris/HCl pH 7.4 at -20°C[13] and this has been our experience with human brain enzyme. In the early studies of the preparation of E. coli DHPR we found that the reductase lost half of its activity upon storage at -70°C. When NADH (20μΜ final concentration) was added to the pooled fractions from the DEAE anion exchange purification step, the light yellow colour had disappeared instantly, but reappeared upon standing in air without loss of enzyme activity. However when DTT (2mM final concentration) was added to the pooled fracions, 30% of enzyme activity was lost. A more detailed investigation showed that unlike with human reductase, 2mM DTT (final concentration) inactivates the enzyme bothat -20°Cand25°Cwith30% and77% lossof activity, respectively, after 24 h. NADH (20μΜ) protects the enzyme from DTTinactivation at both temperatures. The stability of the purified enzyme under various conditions is summarised in Table 2. The mechanism of DTT inactivation is not clear but it is known that DTTcan reduce FAD and can act as a substrate for some flavoproteins[17]. In view of the above results the general practice of adding DTT to stabilize flavin-containing proteins needs to be reconsidered.
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Presence of dihydrofolate reductase activity Dihydropteridine reductase and dihydrofolate reductase reduce two different tautomers of dihydropterin (1 and 3, respectively, in Scheme 1). Generally DHPRs will not use 7,8-dihydrofolate or 7,8-dihydro(3//)-pterins as substrates and DHFRs do not reduce quinonoid 7,8-dihydro-(6//)-pterins. We have confirmed that no reaction occurred when human brain DHPR was added to 7,8-dihydrofolate (118μΜ) in the presence of NADH (ΙΟΟμ,Μ) at pH 7.4; and E. coli DHFR was inactive in the presence of quinonoid 6methyl-7,8-dihydro-(6//)-pterin (ΙΟΟμ,Μ) and NADPH (54μΜ). There was also no detectable DHPR activity in the R-plasmid R67 encoded DHFR[18] which bears no sequence homology to other DHFRs. The present E. coli DHPR was found to have dihydrofolate reductase activity which was confirmed by determining the kinetic parameters with NADH and NADPH separately as nucleotide cofactors (see Table 3). This reductase, like known dihydrofolate reductases^, but unlike dihydropteridine reductases^, has a Km value for NADPH that is less, by one order of magnitude, than that for NADH. Comparison of DHFR-specific activities in cell-free extracts of the DHFR~ mutant D3-157 (2.54 mU/mg protein using NADPH and 7.19 mU/mg using NADH) with those from the parent strain H712 (wildtype; 28.8 mU/mg protein using NADPH and 6.27 mU/mg using NADH) and a DHFR over-producing strain JFM 223 (729 mU/mg protein using NADPH and 24.3 mU/mg usning NADH) were made. Despite the relatively high level of DHFR activity in the DHFR-deficient strain, which is most probably due to the DHPR, this strain was unable to grow in minimal media without folate-dependent end-products, e.g. thymidine^10'2^. This raises the question of the in vivo capacity of the E. coli DHPR to carry out the function of DHFR. To address this question we attempted to clone the E. coli DHPR gene[22] in order to express the reductase in high levels in D3-157 (DHFR~) to see if the strain would now grow in minimal media. Using an expression cloning vector strategy we isolated a gene that codes for a hemoglobinlike protein with DHPR activity which is different from the present 25.7-kDa protein. Enzyme activity stain The basis of the enzyme activity staining method is that tetrahydro- pterins and folate which are the products of DHPR- and DHFR-catalysed reactions can reduce the soluble tetrazolium MTTdye to yield the insoluble blue formazan^23'24!. The rate of formation of the blue colour is dependent on the reduction potential of the products of the enzymic reactions. The acBrought to you by | Purdue University Libraries Authenticated Download Date | 5/23/15 5:31 AM
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S. Vasudevan, B. Paal und W.L.F. Armarego
tivity staining of the native gel clearly shows that human DHPR does not possess DHFR activity whereas E. coli DHFR and E. coli DHPR both stain positively because tetrahydrofolate reduces MTT rapidly. Human DHPR and E. coli DHPR stain positively for DHPR also because tetrahydropterins formed reduced MTT. It became evident that E. coli DHPR can utilise tetrazolium MTT as an artificial electron acceptor in the absence of folates and pterins and produces a blue colour but more slowly than in the above case. This allowed the pterin-independent oxidoreductase activity to be stained blue on the gels. Human DHPR and E. coli DHFR, not being flavoproteins do not reduce MTT in the absence of pterins and folate, and are not stained blue with the oxidoreductase activity stain (Fig. 1). Molecular mass The activity stain gel (Fig. 1) also shows clearly the molecular mass of the active E. coli DHPR is greater than 18.5 kD (molecular mass DHFR) and much less than 50 kDa (molecular mass of active dimeric human DHPR). The activity staining comparisons suggest that the molecular mass of native E. coli DHPR is the same as that of its subunit (25.7 kDa). Gel filtration studies and comparing the elution time of E. coli DHPR with standard proteins in 50mM Tris/HCl pH 7.4 containing 2mM DTTand 20mM NADH indicated that the protein existed as a dimer with a molecular mass of 54 ±2 kDa^. This is conflicting with the data from the activity staining and gel filtration experiments and prompted the determination of the molecular mass by analytical centrifugation. The analytical ultracentrifugation analysis using the meniscus depletion experiment^255 in 50mM Hepes pH 7.4 at 40 k rpm. (20 °C for 8h) showed that the bulk of the protein was concentrated in a region where its molecular mass (calculated) was 25.7 kDa assuming the density of solution was 1.001 g/m/ and the partial specific volume was 0.73 m//g. It is likely that the gel filtration value may have been over-estimated because of retardation brought about by reactivity with DTT. The trifunctionality of E. coli DHPR The purified enzyme has been shown to possess three separate activities, viz pterin-independent NADH oxidoreductase, dihydropteridine reductase and dihydrofolate reductase activities. By using saturating concentrations of substrates i.e. concentrations at which the initial rates are maximal, and the same amounts of enzyme and NADH (ΙΟΟμΜ), the ratio of the three activities pterin-independent oxidoreductase, DHPR and DHFR are 4.3:1.0:0.05, respectively. The amino-acid composition was determined
Vol. 373 (1992)
and when compared with that of the rat and the human DHPR (seeTable 4) there was superficial similarity in composition. The molecular mass (25.7 kDa) was closer to the subunit molecular mass of human DHPR (active as dimer of subunit molecular mass 50 kDa[26)) than the molecular mass of E. coli DHFR (18.5 kDa), but like the latter it is active as a monomer. Attempts to isolate the gene coding for the E. coli reductase by expression cloning using a cosmid library resulted in the discovery of a novel 44 kDa flavo-haemoglobin with dihydropteridine reductase activity[22]. Much is known about the mechanism of reduction of the non-flavin containing DHFR[19] and DHPR[13 271 but the presence of FAD in a protein possessing both these activities led us to examine the effect of the well known irreversible flavin inhibitor, A^TV-dimethylpropargyl amine[28~30] on all three activities. The amine did not inhibit the activities equally (see Materials and Methods). The pterin-independent oxidoreductase activity was weakly inhibited compared with other flavoproteins (e.g. ref.[29]), the DHPR activity immediately decreased by 40% and thereafter was very slowly inhibited, and the DHFR activity was not affected.These data imply that the flavin may not be involved in all three reactions. The presence of all three activities in the same protein prompts the speculation that mammalian dihydrofolate reductases and dihydropteridine reductases may have evolved from the same oxidoreductase flavoprotein. References 1 2 3 4 5 6 7 8 9
Shiman, R. (1985) in Folates and Pterins vol. 2 (Blakley, R.L. & Benkovic, S.J., eds.), pp. 179-249, J. Wiley & Sons, New York. Kaufman, S. & Kaufman, E.E. (1985) in Folates and Pterins vol. 2 (Blakley, R.L. & Benkovic, S.J., eds.), pp. 251-352, J.Wiley & Sons, New York. K hn, D.M. & Lovenberg, W. (1985) in Folates and Pterins vol. 2 (Blakley, R.L. & Benkovic, S.J., eds.), pp. 353-399, J. Wiley & Sons, New York. Armarego, W.L.F. & Kosar-Hashemi, Β. (1989) in Chemistry and Biology of Pteridines (Curtius, H.-C., Gisla, S. & Blau, N., eds.), pp. 620-623,W. de Gruyter, Berlin. Vasudevan, S.G., Shaw, D.C. & Armarego, W.L.F. (1988) Biochem. J. 255, 581-588. Dahl, H.-H.M., Hutchinson,W, McAdam, W.,Wake, S., Morgan, F.J. & Cotton, R.G.H. (1987) Nucl. Acids Res. 15,25-28. Lockyer, J., Cook, R.G., Milstein, S., Kaufmann, S., Woo, S.L.C. & Ledley, F.D. (1987) Proc. Natl. Acad. Sei. USA 84, 3329-3333. Shahbaz, M., Hoch, J.A., Trach, K.A., Hural, J.A., Webber, S. & Whiteley, J.M. (1987) J. Biol. Chem. 262, 16412-16416. Armarego,W.L.F. &Vasudevan, S.G. (1989) in Chemistry and Biology of Pteridines (Curtius, H.-Ch., Gisla, S. & Blau, N., eds.) pp. 616-619.
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Vol. 373 (1992)
E. coli Dihydropteridine Reductase with Dihydrofolate Reductase activity
10 Singer, S., Ferone, R., Walton, L. & Elwell, L. (1985) /. Bacterial 164, 470-472. 11 Smith, D.R., Rood, J.I., Bird, P.I., Sneddon, M.K., Calvo, J.M. & Morrison, J.F. (1982) /. Biol. Chem. 257, 9043-9048. 12 Armarego, W.L.F. & Schou, H. (1978) Aust. J. Chem. 31, 1081-1094. 13 Armarego, W.L.F., Randies, D. & Waring, P. (1984) Med. Res. Rev. 4, 267-321. 14 Cornish-Bowden, A. & Endrenyi, L. (1981) Biochem. J. 193,1005-1006. 15 Cotton, R.G.H. & Jennings, I. (1978) Eur. J. Biochem. 83, 319-324. 16 Kaufman, S. (1961) J. Biol. Chem. 236,804-810. 17 Desa, R.J. (1976) in Flavins and Flavoproteins (Singer, T.P., ed.) pp. 720-725, Elsevier, Amsterdam. 18 Matthews, D.A., Smith, S.L., Baccanari, D.P., Burchall, J.J., Oatley, S.J. & Kraut, J. (1986) Biochemistry 25, 4194-4204. 19 Blakley, R.L. (1984) in Folates and Pterins vol. 1 (Blakley, R.L. & Benkovic, S.J., eds.) pp. 191-253.
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20 Armarego,W.L.F. (1989) Pteridines 1,179-188. 21 Vasudevan, S.G. (1989) Ph.D.Thesis, Australian National University. 22 Vasudevan, S.G., Armarego, W.L.F., Shaw, D.C., Lilley, P.E., Dixon, N.E. & Poole, R.K. (1991) Mol Gen. Genet. 266,49-58. 23 Hooper,WD. (1969) Pure andAppl Chem. 19,221-241. 24 Gunlak, E.G., Neal, G.E. & Williams, D.C. (1966) Biochem. J. 101,29P-30P. 25 Yphantis, D.D. (1964) Biochemistry 3,295-316. 26 Firgaira, F, Cotton, R.G.H. & Danks, D. (1981) Biochem. J. 197, 31-43. 27 Randies, D. (1986) Eur. J. Biochem. 155, 301-304. 28 Gisla, S., Wenz, A. &Thorpe, C. (1980) in Enzyme Inhibitors (Brodbeck, U., ed.) pp. 43-60, Verlag Chemie, Basel. 29 Maycock, A.L., Abeles, R.H., Salach, J.I. & Singer, T.P. (1976) Biochemistry 15,114-125. 30 Abeles, R.H. (1978) in Enzyme-Activated Irreversible Inhibitors (Seiler, N., Jung, M.J. & Koch-Weser, J., eds.) pp. 1-12, Elsevier/North Holland, Amsterdam.
S.G. Vasudevan3, B. Paal and W.L.F. Armarego*b, Research School of Chemistry3 and Protein Biochemistry Group15, Division of Biochemistry and Molecular Biology, The John Curtin School of Medical Research, The Australian National University, G.P.O. Box 4, Canberra City, ACT 2601, Australia. * To whom correspondence should be addressed.
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