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[52] Glucose-6-phosphate dehydrogenase from Pseudomonas W6
346
METHYLOTROPHY
[52] G l u c o s e - 6 - p h o s p h a t e
Dehydrogenase
[52]
from
Pseudomonas W6 B y D I E T M A R M I E T H E a n d W O L F G A N G BABEL
v-Glucose 6-phosphate + NAD(P) + ~.~ D-6-phosphogluconate + NAD(P)H + H +
Glucose-6-phosphate dehydrogenase catalyzes the oxidation of glucose 6-phosphate to 6-phosphogluconate in the presence of NAD + or NADP +. It is involved in the assimilation of formaldehyde via the ketodeoxyphosphogluconate variant of the ribulose monophosphate pathway and in the cyclic oxidation of formaldehyde to CO2 with fibulose monophosphatetype methylotrophs.l The purification and characterization of this enzyme from the obligately methylotrophic Pseudomonas W6 have been the subject of a previous report. 2 The purification and properties of glucose-6phosphate dehydrogenases from Methylomonas M 15,3,4 Pseudornonas C, 5 Methylophilus rnethylotrophus,~ and Methylobacillus flagellatum KT 7 have been reported. Assay Method
Principle. Glucose-6-phosphate dchydrogenasc is measured spectrophotometrically by following the rate of NADH or NADPH formation at 340 nm in the presence of saturating amounts of glucose 6-phosphate and NAD + or NADP +. Reagents Triethylamine (TEA) buffer, 115 raM, pH 7.6 Glucose 6-phosphate, 27.4 m M NAD +, 38.5 m M NADP +, 11.0 m M M g C l 2" 6H20, 55.7 m M Procedure. The assay mixture contains 1.7 ml TEA buffer, 0.1 ml glucose 6-phosphate, 0.1 ml NAD + or NADP +, 50/zl MgCl 2 • 6H20 , and 50/al I C. Anthony, "The Biochemistry of Methylotrophs." Academic Press, London, 1982. 2 D. Miethe and W. Babel, Z. Allg. Mikrobiol. 16, 289 (1976). 3 R. A. Steinbach, H. Sahm, and H. Schiltte, Eur. J. Biochem. 87, 409 (1978). 4 R. A. Steinbach, H. SchQtte, and H. Sahm, this series, Vol. 89, p. 271. 5 A. Ben-Bassat, I. Goldber~ and R. I. Mateles, J. Gen. Microbiol. 116, 213 (1980). 6 A. J. Beardsmore, P. N. G. Aperghis, and J. R. Quayle, J. Gen. Microbiol. 128, 1423 (1982). 7 M. Y. Kiriuchin, L. V. Kletsova, A. Y. Chistoserdov, and Y. D. Tsygankov, F E M S Microbiol. Left. 52, 199 (1988).
METHODS IN ENZYMOLOGY, VOL. 188
Copyright© 1990by AcademicPt¢~, Inc. All rightsof reproductionin any formre~rvcd.
[52]
G6PDH FROM Pseudomonas W6
347
enzyme solution in a total volume of 2.0 ml. Prior to assay, the reaction mixture is brought to 300. NADH or NADPH formation is followed at this temperature in a recording spectrophotometer. The reaction is initiated by the addition of glucose 6-phosphate. The rate of absorbance change is followed at 340 nm for 10 min. Units. One unit of glucose-6-phosphate dehydrogenase activity is defined as that amount of enzyme causing the reduction of 1 gmol of NAD + or NADP + per minute. Specific activity is expressed as units per milligram of protein.
Growth of Bacteria
Pseudomonas W6 is grown aerobically at 30 ° in a mineral medium containing, per liter of doubly distilled water, NH4C1, 1.0 g; KH2PO 4, 0.3 g; MgSO4-7H20, 0.2g; CuSO4.5H20, 24 mg; ZnCI2, 12mg; COSO4"7H20, 3mg; MnSO4-4H20, 3mg; H3PO4, 34mg; FeCI3" 6H20, 2 mg; and 10 ml of methanol. The cells are inoculated into 100 ml of the medium in 500-ml shake flasks and harvested at the end of logarithmic growth phase (after around 16 hr) by centrifugation at 5000 g and 0 - 4 °. The biomass is washed twice with ice-cold water and stored at this temperature until use. Purification P r o c e d u r e All operations are carded out in the cold room at around 4 °. Step 1: Preparation of Crude Extract. The cell paste is suspended in 50 m M TEA buffer, pH 7.6, at 100 nag dry weight per milliliter. The ceils are disrupted by two passages through an Aminco French pressure cell press at 96 MPa. The resulting homogenate is centrifuged at 23,000 g for 20 min at 0 ° to remove unbroken cells and cell debris. The glucose-6phosphate dehydrogenase activity is present in the supernatant solution. Step 2: Protamine Sulfate Treatment. Under mechanical stirring, a 1% solution of protamine sulfate is added dropwise to the crude extract. After an additional 20 min, the precipitate is removed by centrifugation. Step 3: Ammonium Sulfate Fractionation. To the supernatant from Step 2, 43 g of solid ammonium sulfate per 100 ml (65% saturation) is added. The suspension is centrifuged at 20,000 g after stirring for 30 min at 0 °. Another 7 g of ammonium sulfate (75% saturation) is added, and the centrifugation is repeated after stirring for 30 min. The collected precipitate is dissolved in 50 m M TEA buffer, pH 7.6 and dialyzed for 18 hr at 0 - 4 ° against a 200-fold volume of the buffer.
348
METHYLOTROPHY
[52]
Step 4: Chromatography on DEAE-Cellulose. The dialyzed enzyme solution is applied to a DEAE-cellulose column (2.5 × 20 cm) equilibrated with 50 m M T E A buffer, pH 7.6. The column is washed with 500 ml of the same buffer and then with 500 ml of the buffer containing 0.1 M of KCI. Enzyme elution is performed with a linear gradient from 0.1 to 1.0 M KCI in a total volume of 2 liters of TEA buffer. The fractions containing high specific activity are concentrated by ammonium sulfate treatment as described above. The precipitate is dissolved in 50 m M TEA buffer, pH 7.6, and dialyzed for 12 hr at 0 - 4 ° against a 200-fold volume of 50 m M TEA buffer. To the dialyzed extract solid ammonium sulfate is added to a final concentration of 1 M. The dialyzate is stored at - 2 0 °. A typical purification is summarized in Table I. Properties
Purity. The described procedure results in an about 80-fold enrichment with a yield of around 10%. The enzyme preparation is not homogeneous. Polyacrylamide gel electrophoresis of the enzyme solution revealed several bands of protein. However, the enzyme preparation is free of activities that might interfere with the assay of glucose-6-phosphate dehydrogenase. Kinetic Properties. Glucose-6-phosphate dehydrogenase from Pseudomonas W6 catalyzes the oxidation of glucose 6-phosphate with NAD + as well as NADP + as the electron acceptor. The affinity for NADP + is approximately 13 times higher than that for NAD +, but the catalytic activity is higher at greater NAD ÷ concentrations that at saturating NADP + concentrations. The ratio of maximum reaction rates Vm~NAD/Vm~NADpis 1.6. Since the ratio of the specific activities of the NAD + and NADP+-linked reaction does not change in the course of purification, only one enzyme protein appears to be responsible for the catalysis. The Km values in the presence of 1 m M MgC12 are 0.24 m M for NAD + and 0.018 raM for NADP, ÷ and s0.5 values with respect to glucose 6-phosphate are 0.18 and 0.13 mM, respectively. pH Optimum. The enzyme is active in the pH range between 7 and 9 and exhibits maximum activity at around pH 8.8. Stability. The purified enzyme shows no loss of activity over a period of 12 months when stored as a solution of 1 M ammonium sulfate at - 2 0 o. Inhibitors. Glucose-6-phosphate dehydrogenase is a key enzyme in the assimilation of glucose. It controls the flux of hexoses to pentoses or to trioses. Therefore, it is plausible that the activity is regulated by various metabolites. In Pseudomonas W6 this enzyme is involved in the assimilation and dissimilation of methanol, but it does not immediately control the distribution of the carbon flux because glucose 6-phosphate is not a branch point. Therefore, its regulatory properties are of particular interest, and the
d d ~
0
•~ -~ < Z u~ 0 0
D M u. 0 Z <
D
>
0
350
METHYLOTROPHY
[53]
effect of a number of metabolites has been studied. With respect to ATP and NADH inhibition, the Pseudomonas W6 glucose-6-phosphate dehydrogenase resembles those of Hydrogenomonas H 16, s Pseudomonas fluorescens, 9 and Rhodopseudomonas sphaeroides. 1° NADH competitively inhibits only the NAD+-linked reaction; the NADP+-linked oxidation is not influenced by NADH. NADPH does not inhibit the catalysis at all. ATP (1 raM) causes a 37% inhibition of the NAD+-linked glucose-6-phosphate dehydrogenase and a 13% inhibition of the NADP+-linked enzyme. Intermediates of carbon metabolism at a concentration of 2 m M hardly decrease the enzyme activity; only acetyl-coenzyme A at a concentration of 1 m M inhibits the activity (by 50%). s F. Blackkolb and H. G. Schlegel, Arch. Microbiol. 63, 177 (1968). 9 j. Schindler and H. G. Schlegel, Arch. Microbiol. 66, 69 (1969). ~0E. Ohmann, R. Borriss, and K. R. Rindt, Z. Allg. Mikrobiol. 10, 37 (1970).
[53] C i t r a t e S y n t h a s e s f r o m M e t h y l o t r o p h s B y G A B R I E L E M O L L E R - K R A F T a n d W O L F G A N G BABEL Oxaloacctate + acetyl-CoA
, citrate + CoASH
Citrate synthase (EC 4.1.3.7) is the key regulatory enzyme of the amphibolic tricarboxylic acid cycle and is inhibited by energy metabolites.l-3 In methylotrophic nutrition the energy-supplying function of the tricarboxylic acid cycle is dispensable. Assay M e t h o d Principle. Citrate synthasc activity is measured according to the principle described by Srere et al. 4 The CoASH formed reacts with DTNB (5,5'-dithiobis-2-nitrobenzoic acid) giving thionitrobenzoate which adsorbs at 412 nm. Because the citrate synthase is inhibited by DTNB and the oxaloacetate can prevent this inhibition, the reaction is started by the addition of enzyme. Deacylase activity is estimated by omission of oxaloacetate. Reagents
Tris-HC1 buffer, pH 7.6, 0.2 M DTNB, 18 m M i p. D. J. Weitzman and D. Jones, Nature (London) 219, 270 (1968). 2 V. R. Flechtner and R. S. Hanson, Biochim. Biophys. Acta 222, 253 (1970). 3 p. D. J. Weitzman, Soc. Appl. Bacteriol. Syrup. Set. 8, 107 (1980). 4 p. A. Srere, H. Brazil, and L. Gonen, Acta Chem. Scand. (Suppl. 1) 17, 129 (1963).
METHODS IN ENZYMOIX)GY,VOL. 188
Copyright© 1990by AcademicPrem,Inc. All fightsof reproductionin any formreserved.
METHYLOTROPHY
[52] G l u c o s e - 6 - p h o s p h a t e
Dehydrogenase
[52]
from
Pseudomonas W6 B y D I E T M A R M I E T H E a n d W O L F G A N G BABEL
v-Glucose 6-phosphate + NAD(P) + ~.~ D-6-phosphogluconate + NAD(P)H + H +
Glucose-6-phosphate dehydrogenase catalyzes the oxidation of glucose 6-phosphate to 6-phosphogluconate in the presence of NAD + or NADP +. It is involved in the assimilation of formaldehyde via the ketodeoxyphosphogluconate variant of the ribulose monophosphate pathway and in the cyclic oxidation of formaldehyde to CO2 with fibulose monophosphatetype methylotrophs.l The purification and characterization of this enzyme from the obligately methylotrophic Pseudomonas W6 have been the subject of a previous report. 2 The purification and properties of glucose-6phosphate dehydrogenases from Methylomonas M 15,3,4 Pseudornonas C, 5 Methylophilus rnethylotrophus,~ and Methylobacillus flagellatum KT 7 have been reported. Assay Method
Principle. Glucose-6-phosphate dchydrogenasc is measured spectrophotometrically by following the rate of NADH or NADPH formation at 340 nm in the presence of saturating amounts of glucose 6-phosphate and NAD + or NADP +. Reagents Triethylamine (TEA) buffer, 115 raM, pH 7.6 Glucose 6-phosphate, 27.4 m M NAD +, 38.5 m M NADP +, 11.0 m M M g C l 2" 6H20, 55.7 m M Procedure. The assay mixture contains 1.7 ml TEA buffer, 0.1 ml glucose 6-phosphate, 0.1 ml NAD + or NADP +, 50/zl MgCl 2 • 6H20 , and 50/al I C. Anthony, "The Biochemistry of Methylotrophs." Academic Press, London, 1982. 2 D. Miethe and W. Babel, Z. Allg. Mikrobiol. 16, 289 (1976). 3 R. A. Steinbach, H. Sahm, and H. Schiltte, Eur. J. Biochem. 87, 409 (1978). 4 R. A. Steinbach, H. SchQtte, and H. Sahm, this series, Vol. 89, p. 271. 5 A. Ben-Bassat, I. Goldber~ and R. I. Mateles, J. Gen. Microbiol. 116, 213 (1980). 6 A. J. Beardsmore, P. N. G. Aperghis, and J. R. Quayle, J. Gen. Microbiol. 128, 1423 (1982). 7 M. Y. Kiriuchin, L. V. Kletsova, A. Y. Chistoserdov, and Y. D. Tsygankov, F E M S Microbiol. Left. 52, 199 (1988).
METHODS IN ENZYMOLOGY, VOL. 188
Copyright© 1990by AcademicPt¢~, Inc. All rightsof reproductionin any formre~rvcd.
[52]
G6PDH FROM Pseudomonas W6
347
enzyme solution in a total volume of 2.0 ml. Prior to assay, the reaction mixture is brought to 300. NADH or NADPH formation is followed at this temperature in a recording spectrophotometer. The reaction is initiated by the addition of glucose 6-phosphate. The rate of absorbance change is followed at 340 nm for 10 min. Units. One unit of glucose-6-phosphate dehydrogenase activity is defined as that amount of enzyme causing the reduction of 1 gmol of NAD + or NADP + per minute. Specific activity is expressed as units per milligram of protein.
Growth of Bacteria
Pseudomonas W6 is grown aerobically at 30 ° in a mineral medium containing, per liter of doubly distilled water, NH4C1, 1.0 g; KH2PO 4, 0.3 g; MgSO4-7H20, 0.2g; CuSO4.5H20, 24 mg; ZnCI2, 12mg; COSO4"7H20, 3mg; MnSO4-4H20, 3mg; H3PO4, 34mg; FeCI3" 6H20, 2 mg; and 10 ml of methanol. The cells are inoculated into 100 ml of the medium in 500-ml shake flasks and harvested at the end of logarithmic growth phase (after around 16 hr) by centrifugation at 5000 g and 0 - 4 °. The biomass is washed twice with ice-cold water and stored at this temperature until use. Purification P r o c e d u r e All operations are carded out in the cold room at around 4 °. Step 1: Preparation of Crude Extract. The cell paste is suspended in 50 m M TEA buffer, pH 7.6, at 100 nag dry weight per milliliter. The ceils are disrupted by two passages through an Aminco French pressure cell press at 96 MPa. The resulting homogenate is centrifuged at 23,000 g for 20 min at 0 ° to remove unbroken cells and cell debris. The glucose-6phosphate dehydrogenase activity is present in the supernatant solution. Step 2: Protamine Sulfate Treatment. Under mechanical stirring, a 1% solution of protamine sulfate is added dropwise to the crude extract. After an additional 20 min, the precipitate is removed by centrifugation. Step 3: Ammonium Sulfate Fractionation. To the supernatant from Step 2, 43 g of solid ammonium sulfate per 100 ml (65% saturation) is added. The suspension is centrifuged at 20,000 g after stirring for 30 min at 0 °. Another 7 g of ammonium sulfate (75% saturation) is added, and the centrifugation is repeated after stirring for 30 min. The collected precipitate is dissolved in 50 m M TEA buffer, pH 7.6 and dialyzed for 18 hr at 0 - 4 ° against a 200-fold volume of the buffer.
348
METHYLOTROPHY
[52]
Step 4: Chromatography on DEAE-Cellulose. The dialyzed enzyme solution is applied to a DEAE-cellulose column (2.5 × 20 cm) equilibrated with 50 m M T E A buffer, pH 7.6. The column is washed with 500 ml of the same buffer and then with 500 ml of the buffer containing 0.1 M of KCI. Enzyme elution is performed with a linear gradient from 0.1 to 1.0 M KCI in a total volume of 2 liters of TEA buffer. The fractions containing high specific activity are concentrated by ammonium sulfate treatment as described above. The precipitate is dissolved in 50 m M TEA buffer, pH 7.6, and dialyzed for 12 hr at 0 - 4 ° against a 200-fold volume of 50 m M TEA buffer. To the dialyzed extract solid ammonium sulfate is added to a final concentration of 1 M. The dialyzate is stored at - 2 0 °. A typical purification is summarized in Table I. Properties
Purity. The described procedure results in an about 80-fold enrichment with a yield of around 10%. The enzyme preparation is not homogeneous. Polyacrylamide gel electrophoresis of the enzyme solution revealed several bands of protein. However, the enzyme preparation is free of activities that might interfere with the assay of glucose-6-phosphate dehydrogenase. Kinetic Properties. Glucose-6-phosphate dehydrogenase from Pseudomonas W6 catalyzes the oxidation of glucose 6-phosphate with NAD + as well as NADP + as the electron acceptor. The affinity for NADP + is approximately 13 times higher than that for NAD +, but the catalytic activity is higher at greater NAD ÷ concentrations that at saturating NADP + concentrations. The ratio of maximum reaction rates Vm~NAD/Vm~NADpis 1.6. Since the ratio of the specific activities of the NAD + and NADP+-linked reaction does not change in the course of purification, only one enzyme protein appears to be responsible for the catalysis. The Km values in the presence of 1 m M MgC12 are 0.24 m M for NAD + and 0.018 raM for NADP, ÷ and s0.5 values with respect to glucose 6-phosphate are 0.18 and 0.13 mM, respectively. pH Optimum. The enzyme is active in the pH range between 7 and 9 and exhibits maximum activity at around pH 8.8. Stability. The purified enzyme shows no loss of activity over a period of 12 months when stored as a solution of 1 M ammonium sulfate at - 2 0 o. Inhibitors. Glucose-6-phosphate dehydrogenase is a key enzyme in the assimilation of glucose. It controls the flux of hexoses to pentoses or to trioses. Therefore, it is plausible that the activity is regulated by various metabolites. In Pseudomonas W6 this enzyme is involved in the assimilation and dissimilation of methanol, but it does not immediately control the distribution of the carbon flux because glucose 6-phosphate is not a branch point. Therefore, its regulatory properties are of particular interest, and the
d d ~
0
•~ -~ < Z u~ 0 0
D M u. 0 Z <
D
>
0
350
METHYLOTROPHY
[53]
effect of a number of metabolites has been studied. With respect to ATP and NADH inhibition, the Pseudomonas W6 glucose-6-phosphate dehydrogenase resembles those of Hydrogenomonas H 16, s Pseudomonas fluorescens, 9 and Rhodopseudomonas sphaeroides. 1° NADH competitively inhibits only the NAD+-linked reaction; the NADP+-linked oxidation is not influenced by NADH. NADPH does not inhibit the catalysis at all. ATP (1 raM) causes a 37% inhibition of the NAD+-linked glucose-6-phosphate dehydrogenase and a 13% inhibition of the NADP+-linked enzyme. Intermediates of carbon metabolism at a concentration of 2 m M hardly decrease the enzyme activity; only acetyl-coenzyme A at a concentration of 1 m M inhibits the activity (by 50%). s F. Blackkolb and H. G. Schlegel, Arch. Microbiol. 63, 177 (1968). 9 j. Schindler and H. G. Schlegel, Arch. Microbiol. 66, 69 (1969). ~0E. Ohmann, R. Borriss, and K. R. Rindt, Z. Allg. Mikrobiol. 10, 37 (1970).
[53] C i t r a t e S y n t h a s e s f r o m M e t h y l o t r o p h s B y G A B R I E L E M O L L E R - K R A F T a n d W O L F G A N G BABEL Oxaloacctate + acetyl-CoA
, citrate + CoASH
Citrate synthase (EC 4.1.3.7) is the key regulatory enzyme of the amphibolic tricarboxylic acid cycle and is inhibited by energy metabolites.l-3 In methylotrophic nutrition the energy-supplying function of the tricarboxylic acid cycle is dispensable. Assay M e t h o d Principle. Citrate synthasc activity is measured according to the principle described by Srere et al. 4 The CoASH formed reacts with DTNB (5,5'-dithiobis-2-nitrobenzoic acid) giving thionitrobenzoate which adsorbs at 412 nm. Because the citrate synthase is inhibited by DTNB and the oxaloacetate can prevent this inhibition, the reaction is started by the addition of enzyme. Deacylase activity is estimated by omission of oxaloacetate. Reagents
Tris-HC1 buffer, pH 7.6, 0.2 M DTNB, 18 m M i p. D. J. Weitzman and D. Jones, Nature (London) 219, 270 (1968). 2 V. R. Flechtner and R. S. Hanson, Biochim. Biophys. Acta 222, 253 (1970). 3 p. D. J. Weitzman, Soc. Appl. Bacteriol. Syrup. Set. 8, 107 (1980). 4 p. A. Srere, H. Brazil, and L. Gonen, Acta Chem. Scand. (Suppl. 1) 17, 129 (1963).
METHODS IN ENZYMOIX)GY,VOL. 188
Copyright© 1990by AcademicPrem,Inc. All fightsof reproductionin any formreserved.