- Email: [email protected]
[56] Serine hydroxymethyltransferases from Methylobacterium organophilum XX
[56]
365
SERINE H Y D R O X Y M E T H Y L T R A N S F E R A S E
mining the glyoxylate-dependent rate of disappearance of acetyl-CoA, using a modification of the method of Dixon and Kornberg.~°
Reagents Tris-HC1, pH 8.0, 200 mM MgC12, 100 mM Acetyl-CoA, 2 mM Sodium glyoxylate, 20 mM Procedure. The assay mixture contains, in a final volume of 0.9 ml, Tris-HC1, 0.4 ml; MgC12, 30 pl; acetyl-CoA, 20/~1; and varying volumes of extract and water. The endogenous rate of disappearance of acetyl-CoA is measured at 232 nm at 30 °, against a blank containing all the assay components except acetyl-CoA. Then 20/zl sodium glyoxylate is added to start the reaction. The rate of cleavage of acetyl-CoA is linear for only about 2 min after the addition of glyoxylate. Units. One unit is defined as the activity (due to the concerted activity of the two enzymes) which catalyzes the cleavage of 1/tmol acetyl-CoA in 1 min. A AE232 value of 0.1 min-~ is equivalent to 9.35 milliunits. 10 G. H. Dixon and H. L. Kornberg, this series, Vol. 5, p. 633.
[56] S e r i n e H y d r o x y m e t h y l t r a n s f e r a s e s
from
Methylobacterium organophilurn X X By MARY E. LIDSTROM Giycine + NS,N t°-methylene tetrahydrofolate ~ serine + tetmhydrofolate
Serine hydroxymethyltransferase (serine transhydroxymethylase, EC 2.1.2.1) catalyzes the interconversion of serine and glycine using the Ct carrier tetrahydrofolate. In most organisms, the physiological role of this enzyme is to synthesize glycine from serine and generate methylene tetrahydrofolate for the C~ pool) However, in methylotrophic bacteria containing the serine cycle for formaldehyde assimilation, during growth on C~ compounds the enzyme functions to generate serine from glycine and methylene tetrahydrofolate, and it is the enzyme that incorporates C ~units derived from the growth substrate into the serine cycle.2 Methylobacterium strains contain two different serine hydroxymethyltransferases, one induced during growth on C~ compounds, involved in the serine cycle, and 1 S. H. M u d d and G. J. Cantoni, in "Comprehensive Biochemistry" (M. Forlein and E. H. Stolz, eds.), p. 1. Elsevier, Amsterdam, 1964. 2 C. Anthony, "The Biochemistry o f Methylotrophs." Academic Press, London, 1982.
METHODS IN ENZYMOLOGY, VOL. 188
Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.
366
METHYLOTROPHY
[56]
the other present during growth on multicarbon compounds, apparently serving the more common hetcrotrophic function,a,4 The C~-specific enzyme is strongly activated by glyoxylate, whereas the heterotrophic enzyme is largely unaffected. This difference can be used to distinguish between the two activities in both crude cell extracts and purified preparations, a Both enzymes have been purified from Methylobacterium organophilum XX, 3 and the methanol-inducible enzyme has been purified from Hyphomicrobium methylovorum. 5 This chapter describes the purification and characteristics of the two M. organophilum XX enzymes. Assay Methods
Principle. Serine hydroxymcthyltransfcrase can be assayed in the direction of glycine synthesis with a continuous or a discontinuous assay. The continuous assay involves coupling the methylene tetrahydrofolate produced to NADP + reduction using commercially available methylenetetrahydrofolate dchydrogcnase.6 The reduction of NADP + is monitored spcctrophotomctrically. The discontinuous assay involves incubating enzyme with specifically radiolabcled serine and then trapping and counting the radiolabeled C~ unit derived from the serine. This is accomplished via the noncnzymatic exchange reaction between formaldehyde and methylene tctrahydrofolatc, which occurs rapidly under the conditions used. Nonlabeled formaldehyde is used to chase the labeled C~ unit out of the methylene tctrahydrofolate, and the formaldehyde is trapped using dimedon (5,5-dimethyl-l,3-cyclohexadione). The C~-dimedon complex can then be extracted into toluene and counted in a scintillation counter. Although the continuous assay is more convenient, it cannot be used to distinguish between the two enzymes because mcthylenetetrahydrofolate dchydrogcnase is inhibited by glyoxylatc. The discontinuous assay must be used in this case, and for all studies involving effectors. Another disadvantage of the continuous assay is that it is carried out at suboptimal pH, owing to the lower pH optimum for the coupling enzyme, methylcnctetrahydrofolate dchydrogcnase. Therefore, it is not useful for kinetic measurements. A more convenient variant of the discontinuous assay has been described recently which involves binding the radiolabeled methylene tctrahydrofolate to DEAE-cellulose paper. 7 Serine hydroxymethyltransferase can be measured in the direction of serine synthesis by measuring glycine3 M. L O'Connor and R. S. Hanson, J. Bacteriol. 124, 985 0975). 4 T. McNerncy and M. L. O'Connor, AppL Environ. MicrobioL 40, 370 (1980). 5 S. S. Miyazald, S. Told, Y. Izumi, and H. Yamada, Eur. Z Biochem. 148, 786 (1986). 6 j. Hcptinstall and J. R. Quayle, Biochem. J. 117, 563 (1970). 7 A. M. Geller and M. Y. Kotb, Anal. Biochem. 180, 120 (1989).
[56]
SERINE HYDROXYMETHYLTRANSFERASE
367
dependent formaldehyde disappearance,5 an assay that also relies on the nonenzymatic exchange reaction between methylene tetrahydrofolate and formaldehyde. The first two assays noted above are described here.
Procedurefor ContinuousAssay Reagents 0.5 M KPO4 buffer, pH 7.5 0.1 M NADP + 2 m M pyridoxal phosphate 5 m M tetrahydrofolate in 0.1 M 2-mercaptoethanol, stored anaerobically under N 2 o r Ar 5 units methylenetetrahydrofolate dehydrogenase (may not be necessary in crude cell extracts) 1.0 M L-serine Assay. One-tenth milliliter of buffer, l0 #1 NADP +, 10 gl pyridoxal phosphate, 50 gl tetrahydrofolate, the methylenetetrahydrofolate dehydrogenase, and the enzyme sample are added to a cuvette containing water to make a total volume of 0.98 ml. The reaction is started with 20 gl L-serine, and the production of NADPH is followed at 340 nm. Assays are routinely run at 30°.
Procedurefor Discontinuous Assay Reagents 0.1 M bicine buffer, pH 8.5 2 m M pyridoxal phosphate 0.1 M 2-mercaptoethanol 80 m M tetrahydrofolate in 0.1 M 2-mercaptoethanol, stored anaerobically under N2 OL-[3-~4C]Serine (Amersham, 55 mCi/mmol), made to 10 m M with DL-serine and diluted to 105 dpm/gmol (5 X 104 dpm/50 gl) 1.0 M sodium acetate buffer, pH 4.5 0.1 M formaldehyde (made from paraformaldehyde by heating)8 0.4 M dimedon (5,5-dimethyl-l,3-cydohexadione) in 50% (v/v) ethanol Toluene Assay. To a test tube is added 0.25 ml bicine buffer, 50/d pyridoxal phosphate, 50 gl 2-mercaptoethanol, l0 gl tetrahydrofolate, and 25 -50 gl enzyme sample. The test tube is preincubated at 30 ° for 5 min, then 25 #l s M. M. Attwood, this volume [47].
368
METHYLOTROPHY
[56]
[~4C]serine is added to start the reaction. The tube is incubated at 30 ° for 15 min, then the reaction is stopped with 0.5 ml sodium acetate buffer. Two-tenths milliliter formaldehyde and 0.3 ml dimedon are added immediately, and the tubes are mixed. The tubes are then heated for 5 min in a boiling water bath and cooled for 5 min on ice. Five milliliters toluene is added, the tubes are stoppered and vortexed at maximum speed for 30 sec, and the tubes are then centrifuged at 5000 g for 2 min. Three milliliters of the upper phase is removed and counted in a water-absorbing cocktail, such as AquasoMI. Controls are carried out with no enzyme and with mixtures to which the sodium acetate buffer is added before the labeled serine, and the average background readings are subtracted from the total radioactivity. The assay is linear with time for 20-25 min. The decays per minute obtained are converted to micromoles formaldehyde, correcting for the fraction of the toluene sampled, the efficiency of counting, and the efficiency of extraction of the formaldehyde into the dimedon. The latter is approximately 80% using this procedure, but it can be tested directly using [~4C]formaldehyde. The specific activity of the labeled serine must be corrected for the presence of the b-serine, which is assumed to account for 50% of the total. Definition of Units. One unit of serine hydroxymethyltransferase activity is defined as the amount of enzyme required to generate 1 /zmol of product per minute. Specific activity is defined as units per milligram protein. Growth of Methylobacterium organophilurn X X
Methylobacterium organophilum XX (ATCC 27886) is grown at 30 ° in a mineral salts medium on either methanol or succinate, as described for Methylobacterium extorquens AM1 in another chapter in this volume. 9 Cells are harvested, washed once with 50 m M KPO4 buffer, pH 7.3, and frozen at - 2 0 °. Purification of Glyoxylate-Activated (C l - S p e c i f i c ) S e r i n e Hydroxymethyltransferase All purification procedures are performed at 4 ° . All buffers contain 50 n M pyridoxal phosphate, 5 IzM dithiothreitol, and 1 m M EDTA. All buffers also contain 30% (v/v) glycerol unless otherwise noted. For these purifications, the discontinuous assay is used so that the correct isoenzyme may be distinguished. 9 C. Krema and M. E. Lidstrom, this volume [57].
[56]
SERINE HYDROXYMETHYLTRANSFERASE
369
Step 1: Preparation of Cell-Free Extracts. Frozen methanol-grown cells (10-20 g wet weight) are thawed in 20-40 ml ofS0 m M KPO4 buffer, pH 7.3, containing the additions noted above (buffer A) and passed through a French pressure cell 3 times at 110 MPa. The extract is centrifuged at 20,000 g for 20 min, and the supernatant is used for further purification. Magnesium chloride (1 mM), ribonuclease (50/~g/ml), and deoxyribonuclease (50/~g/ml) are added, and the preparation is dialyzed overnight at 4 o against 3 changes of buffer A plus 1 m M MgC12. Step 2: DEAE-Cellulose Column Chromatography 1. A DEAE-cellulose (Whatman DE-52) column (2 × 10 cm) is prepared and equilibrated with buffer A. The enzyme preparation from Step 1 is layered onto the top and eluted with buffer A at a flow rate of 0.5 ml/min. The enzyme elutes with the buffer. Step 3: DEAE-Cellulose Column Chromatography 2. The fractions from Step 2 containing the highest specific activity are pooled, diluted 5-fold with distilled water, and layered onto a second DEAE column (2 × 10 cm) equilibrated with 10 m M KPO4 buffer, pH 7.3, containing the same additions as buffer A (buffer B). The column is washed with 50 ml buffer B, and the enzyme is eluted using a linear potassium acetate gradient (0 to 0.25 M in buffer B). The tubes containing the highest specific activity are pooled and concentrated using an Amicon Diaflo ultrafiltration unit (Danvers, MA) equipped with an XM50 membrane. Step 4: Preparative Electrophoresis. The concentrated enzyme preparation from Step 3 is layered onto a Sephadex G-25 column which has been equilibrated with 60 m M Tris-HC1 buffer (pH 8.0; no glycerol; buffer C) and which has been prepared in a stoppered Btichler Fractophorator column. The top of the column is sealed with a 1-cm layer of 0.5% (w/v) agar, and the column is inserted into the Fractophorator apparatus. Electrophoresis is performed at 10 mA with buffer C. After 20 to 34 hr, the enzyme band is discernible near the center of the column. The current is then turned off, the agar seal is broken, and the band is eluted with buffer C. The fractions containing the highest specific activity are pooled and stored at -20°. Purification of Glyoxylate-Insensitive (Heterotrophic) Serine Hydroxymethyltransferase
Step 1: Preparation of Cell-Free Extracts. Cell free extracts are prepared as described for the glyoxylate-activated enzyme, except that succinategrown cells are used. All buffers include the additions noted previously. Step 2: DEAE-Cellulose Column Chromatography 1. The dialyzed extract is loaded onto a DEAE-cellulose column prepared as described for the
370
METHYLOTROPHY
[56]
glyoxylate-activated enzyme. About 10% of the total activity flutes with the buffer, and this activity is activated by glyoxylate. The remainder of the activity is then batch fluted with 0.2 M potassium acetate in buffer A. This activity is glyoxylate-insensitive. The fractions of the glyoxylate-insensitive enzyme containing the highest activity are pooled and dialyzed against 3 changes of 10 m M Tris-citrate buffer (pH 7.8; buffer D). Step 3: DEAE-Cellulose Column Chromatography 2. The dialyzed preparation from Step 2 is loaded onto a second DEAE-cellulose column (2 × 10 cm) which has been equilibrated with buffer D. The column is washed with 50 ml buffer D, then 50 ml buffer D containing 0.1 M potassium acetate. The enzyme is fluted with buffer D containing 0.2 M potassium acetate. Fractions containing the highest specific activity are pooled and concentrated as noted above. Step 4: Glycerol Density Gradient. A linear 10 to 300/0 (v/v) glycerol density gradient is prepared in polyallomer tubes and precooled to 4 °. Samples (0.5 ml) of the concentrated preparation from Step 3 are layered onto each tube and the tubes are centrifuged at 38,000 rpm for 24 hr at 4* in a type SW41 rotor in a Beckman Model L-2 centrifuge. The tubes are punctured with an 18-gauge needle and 12-drop fractions are collected. The fractions containing the highest specific activity are pooled and stored at - 2 0 °. The purification of both enzymes is summarized in Table I. 3 Properties Both serine hydroxymethyltransferase enzymes are present in cells grown on methanol and on succinate, but the major isoenzyme in each case represents about 90% of the total activity. 3 These are separated on the first DEAE-cellulose column. A summary of the properties of each enzyme is presented in Table II. Purity and Stability. Both enzymes are pure as judged by denaturing and nondenaturing polyacrylamide gel electrophoresis, staining with Coomassie blue. A minor band is observed on native gels in the case of the glyoxylate-insensitive enzyme, but it shows activity when gels are sliced and assayed. The glyoxylate-activated enzyme is stable at - 2 0 °, showing no decrease in activity after 2 months, and also shows no decrease in activity after 24 hr at 4 °. The glyoxylate-insensitive enzyme is less stable, however, and shows a 50% reduction in activity after storage for 2 months at - 2 0 ° and an 80% loss of activity after 24 hr at 4 °. Kinetic Properties. Both enzymes show similar apparent Km and V~, values, as determined with the pooled fractions from the second DEAEcellulose column in both cases. These are 1.25 m M and 0.1 gmol/min/mg protein for the glyoxylate-activated enzyme and 1.0 m M and 85 nmol/
[56]
SERINE HYDROXYMETHYLTRANSFERASE
371
TABLE I PURIFICATION OF TWO SERINE HYDROXYMETHYLTRANSFERASE ENZYMES FROM
Methylobacterium organophilumXX
Step Methanol-grown cells 1. Ceil-frce extract 2. DEAE 1 3. DEAE 2 4. Electrophoresis Suecinate-grown cells 1. Crude extract 2. DEAE 1 3. DEAE 2 4. Glycerol gradient
Total protein (mg)
Total units
Specific activity
Yield (%)
Purification -fold
366 222 69 2
15.8 9.3 3.7 1.2
0.043 0.045 0.053 0.6
100 59 23 7.6
-1.04 1.23 11.2
430 66 32 2
9.7 4.2 2.2 1.0
0.023 0.043 0.069 0.52
100 44 23 9.4
-1.9 3.1 23.0
min/mg protein for the glyoxylate-insensitive enzyme. pH Optimum. The pH optimum for the glyoxylate-activated enzyme is 8.7-8.8; for the glyoxylate-insensitive enzyme the pH optimum is slightly lower, 8.5 - 8.6. CofactorRequirements. Both enzymes require pyridoxal phosphate and tetrahydrofolate as cofactors. Maximum activity is obtained for both enzymes at 20 gM pyridoxal phosphate and 200 a M tetrahydrofolate? Molecular Weight. The molecular weight of the glyoxylate-stimulated TABLE II PROPERTIES OF SERINE HYDROXYMETHYLTRANSFERASESFROM Methylobacterium
organophilumXX Enzyme
Property
Glyoxylate-activated (C :specific)
Glyoxylate-insensitive (heterotrophic)
Km (raM) V.~ ~mol/min/mg protein) pH optimum Stimulation by cations (1 mM)
1.25 0.10 8.7- 8.8 Ca 2+, K +, Na +
1.00 0.085 8.5- 8.6 Ca 2+, K +, Na +, Mg2+,
Effect ofglyoxylate (5 raM) (% of control) Molecular weight Total Subunit
450
90
200,000 50,000
I00,000 100,000
Mn2+, Zn 2+
372
METnVLOTROPHV
[56]
enzyme is 200,000, as determined by polyacrylamide gel electrophoresis at varying acrylamide concentrations, and by sucrose density centrifugation. One band of 50,000 is found on gels containing sodium dodecyl sulfate (SDS), suggesting that the enzyme is a tetramer. The molecular weight of the glyoxylate-insensitive enzyme on both SDS-containing and native polyacrylamide gels is 100,000, suggesting that it is a monomeric enzyme. Cation Stimulation. Both enzymes are stimulated by 1 m M Ca 2+, K +, and Na +, but only the glyoxylate-insensitive enzyme is stimulated by 1 m M Mg2+, Mn 2+, and Zn2+. 3 Stimulation by Small Molecules. A variety of small molecules have been tested for their effects on activity of both enzymes, and in the case of AMP, ATP, methionine, S-adenosylmethionine, phosphoenolpyruvate, guanine, and thymine, little effect is observed. However, glyoxylate stimulates the glyoxylate-activated enzyme 4.5-fold, whereas it has little effect on the glyoxylate-stimulated enzyme. Glycine inhibits both enzymes about 50%. All molecules were tested at a concentration of 5 mM, except guanine (0.25 m M ) and thymine (1 raM). Serine H y d r o x y m e t h y l t r a n s f e r a s e s in Other Serine Cycle Methylotrophs Although both isoenzymes have been purified from only one serine cycle methylotroph, it is possible to assess the presence of the glyoxylateactivated enzyme in cell-free extracts. In this case, the activation is usually 1- to 2-fold. 4 In Methylobacterium 3A2 and M. extorquens AMI, a glyoxylate-activated activity is present in methanol-grown cells but not in cells grown on multicarbon compounds, 4 suggesting that isoenzymes of serine hydroxymethyltransferase are probably widespread in these bacteria. However, in Hyphomicrobium X and 1t. methylovorum, bacteria that grow only on C~ and Cz compounds, current evidence suggests that only the glyoxylate-activated enzyme is present. The enzyme that has been purified from methanol-grown cells of H. methylovorum is a dimer of 50,000-MW subunits, and antisera prepared against that enzyme does not cross-react with cell-free extracts of M. organophilum XX, 1° suggesting that these enzymes are different.
~0S. S. Miyazaki,S. Told, Y. Izumi,and H. Yamada,Arch.Microbiol.147, 328 (1987).
365
SERINE H Y D R O X Y M E T H Y L T R A N S F E R A S E
mining the glyoxylate-dependent rate of disappearance of acetyl-CoA, using a modification of the method of Dixon and Kornberg.~°
Reagents Tris-HC1, pH 8.0, 200 mM MgC12, 100 mM Acetyl-CoA, 2 mM Sodium glyoxylate, 20 mM Procedure. The assay mixture contains, in a final volume of 0.9 ml, Tris-HC1, 0.4 ml; MgC12, 30 pl; acetyl-CoA, 20/~1; and varying volumes of extract and water. The endogenous rate of disappearance of acetyl-CoA is measured at 232 nm at 30 °, against a blank containing all the assay components except acetyl-CoA. Then 20/zl sodium glyoxylate is added to start the reaction. The rate of cleavage of acetyl-CoA is linear for only about 2 min after the addition of glyoxylate. Units. One unit is defined as the activity (due to the concerted activity of the two enzymes) which catalyzes the cleavage of 1/tmol acetyl-CoA in 1 min. A AE232 value of 0.1 min-~ is equivalent to 9.35 milliunits. 10 G. H. Dixon and H. L. Kornberg, this series, Vol. 5, p. 633.
[56] S e r i n e H y d r o x y m e t h y l t r a n s f e r a s e s
from
Methylobacterium organophilurn X X By MARY E. LIDSTROM Giycine + NS,N t°-methylene tetrahydrofolate ~ serine + tetmhydrofolate
Serine hydroxymethyltransferase (serine transhydroxymethylase, EC 2.1.2.1) catalyzes the interconversion of serine and glycine using the Ct carrier tetrahydrofolate. In most organisms, the physiological role of this enzyme is to synthesize glycine from serine and generate methylene tetrahydrofolate for the C~ pool) However, in methylotrophic bacteria containing the serine cycle for formaldehyde assimilation, during growth on C~ compounds the enzyme functions to generate serine from glycine and methylene tetrahydrofolate, and it is the enzyme that incorporates C ~units derived from the growth substrate into the serine cycle.2 Methylobacterium strains contain two different serine hydroxymethyltransferases, one induced during growth on C~ compounds, involved in the serine cycle, and 1 S. H. M u d d and G. J. Cantoni, in "Comprehensive Biochemistry" (M. Forlein and E. H. Stolz, eds.), p. 1. Elsevier, Amsterdam, 1964. 2 C. Anthony, "The Biochemistry o f Methylotrophs." Academic Press, London, 1982.
METHODS IN ENZYMOLOGY, VOL. 188
Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.
366
METHYLOTROPHY
[56]
the other present during growth on multicarbon compounds, apparently serving the more common hetcrotrophic function,a,4 The C~-specific enzyme is strongly activated by glyoxylate, whereas the heterotrophic enzyme is largely unaffected. This difference can be used to distinguish between the two activities in both crude cell extracts and purified preparations, a Both enzymes have been purified from Methylobacterium organophilum XX, 3 and the methanol-inducible enzyme has been purified from Hyphomicrobium methylovorum. 5 This chapter describes the purification and characteristics of the two M. organophilum XX enzymes. Assay Methods
Principle. Serine hydroxymcthyltransfcrase can be assayed in the direction of glycine synthesis with a continuous or a discontinuous assay. The continuous assay involves coupling the methylene tetrahydrofolate produced to NADP + reduction using commercially available methylenetetrahydrofolate dchydrogcnase.6 The reduction of NADP + is monitored spcctrophotomctrically. The discontinuous assay involves incubating enzyme with specifically radiolabcled serine and then trapping and counting the radiolabeled C~ unit derived from the serine. This is accomplished via the noncnzymatic exchange reaction between formaldehyde and methylene tctrahydrofolatc, which occurs rapidly under the conditions used. Nonlabeled formaldehyde is used to chase the labeled C~ unit out of the methylene tctrahydrofolate, and the formaldehyde is trapped using dimedon (5,5-dimethyl-l,3-cyclohexadione). The C~-dimedon complex can then be extracted into toluene and counted in a scintillation counter. Although the continuous assay is more convenient, it cannot be used to distinguish between the two enzymes because mcthylenetetrahydrofolate dchydrogcnase is inhibited by glyoxylatc. The discontinuous assay must be used in this case, and for all studies involving effectors. Another disadvantage of the continuous assay is that it is carried out at suboptimal pH, owing to the lower pH optimum for the coupling enzyme, methylcnctetrahydrofolate dchydrogcnase. Therefore, it is not useful for kinetic measurements. A more convenient variant of the discontinuous assay has been described recently which involves binding the radiolabeled methylene tctrahydrofolate to DEAE-cellulose paper. 7 Serine hydroxymethyltransferase can be measured in the direction of serine synthesis by measuring glycine3 M. L O'Connor and R. S. Hanson, J. Bacteriol. 124, 985 0975). 4 T. McNerncy and M. L. O'Connor, AppL Environ. MicrobioL 40, 370 (1980). 5 S. S. Miyazald, S. Told, Y. Izumi, and H. Yamada, Eur. Z Biochem. 148, 786 (1986). 6 j. Hcptinstall and J. R. Quayle, Biochem. J. 117, 563 (1970). 7 A. M. Geller and M. Y. Kotb, Anal. Biochem. 180, 120 (1989).
[56]
SERINE HYDROXYMETHYLTRANSFERASE
367
dependent formaldehyde disappearance,5 an assay that also relies on the nonenzymatic exchange reaction between methylene tetrahydrofolate and formaldehyde. The first two assays noted above are described here.
Procedurefor ContinuousAssay Reagents 0.5 M KPO4 buffer, pH 7.5 0.1 M NADP + 2 m M pyridoxal phosphate 5 m M tetrahydrofolate in 0.1 M 2-mercaptoethanol, stored anaerobically under N 2 o r Ar 5 units methylenetetrahydrofolate dehydrogenase (may not be necessary in crude cell extracts) 1.0 M L-serine Assay. One-tenth milliliter of buffer, l0 #1 NADP +, 10 gl pyridoxal phosphate, 50 gl tetrahydrofolate, the methylenetetrahydrofolate dehydrogenase, and the enzyme sample are added to a cuvette containing water to make a total volume of 0.98 ml. The reaction is started with 20 gl L-serine, and the production of NADPH is followed at 340 nm. Assays are routinely run at 30°.
Procedurefor Discontinuous Assay Reagents 0.1 M bicine buffer, pH 8.5 2 m M pyridoxal phosphate 0.1 M 2-mercaptoethanol 80 m M tetrahydrofolate in 0.1 M 2-mercaptoethanol, stored anaerobically under N2 OL-[3-~4C]Serine (Amersham, 55 mCi/mmol), made to 10 m M with DL-serine and diluted to 105 dpm/gmol (5 X 104 dpm/50 gl) 1.0 M sodium acetate buffer, pH 4.5 0.1 M formaldehyde (made from paraformaldehyde by heating)8 0.4 M dimedon (5,5-dimethyl-l,3-cydohexadione) in 50% (v/v) ethanol Toluene Assay. To a test tube is added 0.25 ml bicine buffer, 50/d pyridoxal phosphate, 50 gl 2-mercaptoethanol, l0 gl tetrahydrofolate, and 25 -50 gl enzyme sample. The test tube is preincubated at 30 ° for 5 min, then 25 #l s M. M. Attwood, this volume [47].
368
METHYLOTROPHY
[56]
[~4C]serine is added to start the reaction. The tube is incubated at 30 ° for 15 min, then the reaction is stopped with 0.5 ml sodium acetate buffer. Two-tenths milliliter formaldehyde and 0.3 ml dimedon are added immediately, and the tubes are mixed. The tubes are then heated for 5 min in a boiling water bath and cooled for 5 min on ice. Five milliliters toluene is added, the tubes are stoppered and vortexed at maximum speed for 30 sec, and the tubes are then centrifuged at 5000 g for 2 min. Three milliliters of the upper phase is removed and counted in a water-absorbing cocktail, such as AquasoMI. Controls are carried out with no enzyme and with mixtures to which the sodium acetate buffer is added before the labeled serine, and the average background readings are subtracted from the total radioactivity. The assay is linear with time for 20-25 min. The decays per minute obtained are converted to micromoles formaldehyde, correcting for the fraction of the toluene sampled, the efficiency of counting, and the efficiency of extraction of the formaldehyde into the dimedon. The latter is approximately 80% using this procedure, but it can be tested directly using [~4C]formaldehyde. The specific activity of the labeled serine must be corrected for the presence of the b-serine, which is assumed to account for 50% of the total. Definition of Units. One unit of serine hydroxymethyltransferase activity is defined as the amount of enzyme required to generate 1 /zmol of product per minute. Specific activity is defined as units per milligram protein. Growth of Methylobacterium organophilurn X X
Methylobacterium organophilum XX (ATCC 27886) is grown at 30 ° in a mineral salts medium on either methanol or succinate, as described for Methylobacterium extorquens AM1 in another chapter in this volume. 9 Cells are harvested, washed once with 50 m M KPO4 buffer, pH 7.3, and frozen at - 2 0 °. Purification of Glyoxylate-Activated (C l - S p e c i f i c ) S e r i n e Hydroxymethyltransferase All purification procedures are performed at 4 ° . All buffers contain 50 n M pyridoxal phosphate, 5 IzM dithiothreitol, and 1 m M EDTA. All buffers also contain 30% (v/v) glycerol unless otherwise noted. For these purifications, the discontinuous assay is used so that the correct isoenzyme may be distinguished. 9 C. Krema and M. E. Lidstrom, this volume [57].
[56]
SERINE HYDROXYMETHYLTRANSFERASE
369
Step 1: Preparation of Cell-Free Extracts. Frozen methanol-grown cells (10-20 g wet weight) are thawed in 20-40 ml ofS0 m M KPO4 buffer, pH 7.3, containing the additions noted above (buffer A) and passed through a French pressure cell 3 times at 110 MPa. The extract is centrifuged at 20,000 g for 20 min, and the supernatant is used for further purification. Magnesium chloride (1 mM), ribonuclease (50/~g/ml), and deoxyribonuclease (50/~g/ml) are added, and the preparation is dialyzed overnight at 4 o against 3 changes of buffer A plus 1 m M MgC12. Step 2: DEAE-Cellulose Column Chromatography 1. A DEAE-cellulose (Whatman DE-52) column (2 × 10 cm) is prepared and equilibrated with buffer A. The enzyme preparation from Step 1 is layered onto the top and eluted with buffer A at a flow rate of 0.5 ml/min. The enzyme elutes with the buffer. Step 3: DEAE-Cellulose Column Chromatography 2. The fractions from Step 2 containing the highest specific activity are pooled, diluted 5-fold with distilled water, and layered onto a second DEAE column (2 × 10 cm) equilibrated with 10 m M KPO4 buffer, pH 7.3, containing the same additions as buffer A (buffer B). The column is washed with 50 ml buffer B, and the enzyme is eluted using a linear potassium acetate gradient (0 to 0.25 M in buffer B). The tubes containing the highest specific activity are pooled and concentrated using an Amicon Diaflo ultrafiltration unit (Danvers, MA) equipped with an XM50 membrane. Step 4: Preparative Electrophoresis. The concentrated enzyme preparation from Step 3 is layered onto a Sephadex G-25 column which has been equilibrated with 60 m M Tris-HC1 buffer (pH 8.0; no glycerol; buffer C) and which has been prepared in a stoppered Btichler Fractophorator column. The top of the column is sealed with a 1-cm layer of 0.5% (w/v) agar, and the column is inserted into the Fractophorator apparatus. Electrophoresis is performed at 10 mA with buffer C. After 20 to 34 hr, the enzyme band is discernible near the center of the column. The current is then turned off, the agar seal is broken, and the band is eluted with buffer C. The fractions containing the highest specific activity are pooled and stored at -20°. Purification of Glyoxylate-Insensitive (Heterotrophic) Serine Hydroxymethyltransferase
Step 1: Preparation of Cell-Free Extracts. Cell free extracts are prepared as described for the glyoxylate-activated enzyme, except that succinategrown cells are used. All buffers include the additions noted previously. Step 2: DEAE-Cellulose Column Chromatography 1. The dialyzed extract is loaded onto a DEAE-cellulose column prepared as described for the
370
METHYLOTROPHY
[56]
glyoxylate-activated enzyme. About 10% of the total activity flutes with the buffer, and this activity is activated by glyoxylate. The remainder of the activity is then batch fluted with 0.2 M potassium acetate in buffer A. This activity is glyoxylate-insensitive. The fractions of the glyoxylate-insensitive enzyme containing the highest activity are pooled and dialyzed against 3 changes of 10 m M Tris-citrate buffer (pH 7.8; buffer D). Step 3: DEAE-Cellulose Column Chromatography 2. The dialyzed preparation from Step 2 is loaded onto a second DEAE-cellulose column (2 × 10 cm) which has been equilibrated with buffer D. The column is washed with 50 ml buffer D, then 50 ml buffer D containing 0.1 M potassium acetate. The enzyme is fluted with buffer D containing 0.2 M potassium acetate. Fractions containing the highest specific activity are pooled and concentrated as noted above. Step 4: Glycerol Density Gradient. A linear 10 to 300/0 (v/v) glycerol density gradient is prepared in polyallomer tubes and precooled to 4 °. Samples (0.5 ml) of the concentrated preparation from Step 3 are layered onto each tube and the tubes are centrifuged at 38,000 rpm for 24 hr at 4* in a type SW41 rotor in a Beckman Model L-2 centrifuge. The tubes are punctured with an 18-gauge needle and 12-drop fractions are collected. The fractions containing the highest specific activity are pooled and stored at - 2 0 °. The purification of both enzymes is summarized in Table I. 3 Properties Both serine hydroxymethyltransferase enzymes are present in cells grown on methanol and on succinate, but the major isoenzyme in each case represents about 90% of the total activity. 3 These are separated on the first DEAE-cellulose column. A summary of the properties of each enzyme is presented in Table II. Purity and Stability. Both enzymes are pure as judged by denaturing and nondenaturing polyacrylamide gel electrophoresis, staining with Coomassie blue. A minor band is observed on native gels in the case of the glyoxylate-insensitive enzyme, but it shows activity when gels are sliced and assayed. The glyoxylate-activated enzyme is stable at - 2 0 °, showing no decrease in activity after 2 months, and also shows no decrease in activity after 24 hr at 4 °. The glyoxylate-insensitive enzyme is less stable, however, and shows a 50% reduction in activity after storage for 2 months at - 2 0 ° and an 80% loss of activity after 24 hr at 4 °. Kinetic Properties. Both enzymes show similar apparent Km and V~, values, as determined with the pooled fractions from the second DEAEcellulose column in both cases. These are 1.25 m M and 0.1 gmol/min/mg protein for the glyoxylate-activated enzyme and 1.0 m M and 85 nmol/
[56]
SERINE HYDROXYMETHYLTRANSFERASE
371
TABLE I PURIFICATION OF TWO SERINE HYDROXYMETHYLTRANSFERASE ENZYMES FROM
Methylobacterium organophilumXX
Step Methanol-grown cells 1. Ceil-frce extract 2. DEAE 1 3. DEAE 2 4. Electrophoresis Suecinate-grown cells 1. Crude extract 2. DEAE 1 3. DEAE 2 4. Glycerol gradient
Total protein (mg)
Total units
Specific activity
Yield (%)
Purification -fold
366 222 69 2
15.8 9.3 3.7 1.2
0.043 0.045 0.053 0.6
100 59 23 7.6
-1.04 1.23 11.2
430 66 32 2
9.7 4.2 2.2 1.0
0.023 0.043 0.069 0.52
100 44 23 9.4
-1.9 3.1 23.0
min/mg protein for the glyoxylate-insensitive enzyme. pH Optimum. The pH optimum for the glyoxylate-activated enzyme is 8.7-8.8; for the glyoxylate-insensitive enzyme the pH optimum is slightly lower, 8.5 - 8.6. CofactorRequirements. Both enzymes require pyridoxal phosphate and tetrahydrofolate as cofactors. Maximum activity is obtained for both enzymes at 20 gM pyridoxal phosphate and 200 a M tetrahydrofolate? Molecular Weight. The molecular weight of the glyoxylate-stimulated TABLE II PROPERTIES OF SERINE HYDROXYMETHYLTRANSFERASESFROM Methylobacterium
organophilumXX Enzyme
Property
Glyoxylate-activated (C :specific)
Glyoxylate-insensitive (heterotrophic)
Km (raM) V.~ ~mol/min/mg protein) pH optimum Stimulation by cations (1 mM)
1.25 0.10 8.7- 8.8 Ca 2+, K +, Na +
1.00 0.085 8.5- 8.6 Ca 2+, K +, Na +, Mg2+,
Effect ofglyoxylate (5 raM) (% of control) Molecular weight Total Subunit
450
90
200,000 50,000
I00,000 100,000
Mn2+, Zn 2+
372
METnVLOTROPHV
[56]
enzyme is 200,000, as determined by polyacrylamide gel electrophoresis at varying acrylamide concentrations, and by sucrose density centrifugation. One band of 50,000 is found on gels containing sodium dodecyl sulfate (SDS), suggesting that the enzyme is a tetramer. The molecular weight of the glyoxylate-insensitive enzyme on both SDS-containing and native polyacrylamide gels is 100,000, suggesting that it is a monomeric enzyme. Cation Stimulation. Both enzymes are stimulated by 1 m M Ca 2+, K +, and Na +, but only the glyoxylate-insensitive enzyme is stimulated by 1 m M Mg2+, Mn 2+, and Zn2+. 3 Stimulation by Small Molecules. A variety of small molecules have been tested for their effects on activity of both enzymes, and in the case of AMP, ATP, methionine, S-adenosylmethionine, phosphoenolpyruvate, guanine, and thymine, little effect is observed. However, glyoxylate stimulates the glyoxylate-activated enzyme 4.5-fold, whereas it has little effect on the glyoxylate-stimulated enzyme. Glycine inhibits both enzymes about 50%. All molecules were tested at a concentration of 5 mM, except guanine (0.25 m M ) and thymine (1 raM). Serine H y d r o x y m e t h y l t r a n s f e r a s e s in Other Serine Cycle Methylotrophs Although both isoenzymes have been purified from only one serine cycle methylotroph, it is possible to assess the presence of the glyoxylateactivated enzyme in cell-free extracts. In this case, the activation is usually 1- to 2-fold. 4 In Methylobacterium 3A2 and M. extorquens AMI, a glyoxylate-activated activity is present in methanol-grown cells but not in cells grown on multicarbon compounds, 4 suggesting that isoenzymes of serine hydroxymethyltransferase are probably widespread in these bacteria. However, in Hyphomicrobium X and 1t. methylovorum, bacteria that grow only on C~ and Cz compounds, current evidence suggests that only the glyoxylate-activated enzyme is present. The enzyme that has been purified from methanol-grown cells of H. methylovorum is a dimer of 50,000-MW subunits, and antisera prepared against that enzyme does not cross-react with cell-free extracts of M. organophilum XX, 1° suggesting that these enzymes are different.
~0S. S. Miyazaki,S. Told, Y. Izumi,and H. Yamada,Arch.Microbiol.147, 328 (1987).