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[39] Methylamine dehydrogenase from methylobacillus flagellatum
MADH FROM M. flagellatum
[39]
[39] M e t h y l a m i n e
Dehydrogenase
247
f r o m Methylobacillus
flagellatum By MICnA~.L Y. KmIUKmN, ANDREY Y. CI-nSTOSERDOV,and Y U R I D . TSYGANKOV
Methylamine + pyrroloquinolinc quinonc + H20 "-~ formaldehyde + reduced pyrroloquinolinc quinone
Methylamine dehydrogenase catalyzes the oxidation of methylamine to formaldehyde and ammonia in the presence of an artificial electron acceptor. The enzyme is induced during growth of the bacterium on mcthylamine. Methylamine dehydrogenases have been purified from various methylotrophic bacteria.t-5 Assay Method
Principle. The assay method involves the continuous measurement of the reduction of the artificial electron acceptor phenazine methosulfate (PMS)?2,6-Dichlorophenolindophenol (DCPIP) is used as a redox indicator. The molar extinction coefficient for DCPIP is taken from the work of Armstrong.7 Reagents
Potassium phosphate buffer, 200 raM, pH 7.5 Phenazine methosulfate, 10 m M 2,6-Dichlorophenolindophenol, 1 m M Methylamine hydrochloride, 200 m M Procedure. The assay mixture contains, in a final volume of 1 ml, potassium phosphate buffer, 0.5 ml; water, 0.19 ml; DCPIP, 0.1 ml; PMS, 0.1 ml; cell extract, 10 gl. The mixture is incubated for 5 min at 30 °. After preincubation 0.1 ml of methylamine hydrochloride is added, and the rate of decrease in absorbance is followed at 600 nm. 1 C. Anthony, "The Biochemistry of Methylotrophs." Academic Press, London, 1982. 2 M. Husain and V. Davidson, J. Bacteriol. 169, 1712 (1987). 3 W. C. Kenny and W. McIntir¢, Biochemistry 22, 3858 (1983). 4 G. W. Heywood, N. S. Janschke, P. J. Large, and J. M. Wallis, FEMS Microbiol. Lett. 15, 79 (1982). 5 F. M. D. VeUieux, M. D. J. Frank, M. B. A. Swarte, H. Groendijk, J. A. Duine, J. Drenth, and W. G. J. Hol, Eur. J. Biochem. 154, 383 (1986). 6 R. R. Eady and P. J. Large, Biochem. J. 106, 245 (1968). 7 j. Armstrong, Biochim. Biophys. Acta 86, 194 (1964).
METHODS IN ENZYMOLOGY, VOL. 188
English translation copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
248
METHYLOTROPHY
[39]
Units. One unit of methylamine dehydrogenase is defined as that amount of enzyme catalyzing reduction of 1/~mol of DCPIP per minute at 30 °. Specific activity is expressed as units per milligram of protein. Purification Procedure
Growth of Organism. The strain of Methylobacillus flagellatum s is grown at 42° in a fermentor in a mineral salts medium containing, per liter: Na2HPO4, 6 g; KH2PO4, 3 g; NaC1, 0.5 g; NH4C1, 1 g; CaC12, 0.11 g; MgSO4.7H20, 0.5 g; and methylamine, 3 g. After 72 hr of growth, the culture is harvested by~centrifugation at 3500 g for 60 min, and the pellet is rinsed with cooled 100 m M potassium phosphate buffer, pH 7.0. The cell paste is stored at - 2 0 °. Step 1: Preparation of Cell-Free Extract. Cell paste (30 g wet weight) is suspended in 150 ml of 100 m M Tris-HC1 buffer, pH 8.0. The suspension is disrupted by ultrasonication for 5 rain (10 times, 30 sec each) at 0 ° in an MSE-150 ultrasonic disintegrator and centrifuged at 25,000 g for 60 min, yielding the cell-free extract as supernatant. Step 2: Heat Treatment. The crude extract is heated at 75 ° for 20 min with slow mixing. This suspension is cooled to 5 ° and centrifuged at 25,000 g for 20 min. The supernatant is used further. Step 3: Ammonium Sulfate Fractionation. To the supernatant, solid (NH4)2SO4 is added with stirring up to 70% of saturation and kept at 4 ° for 1 hr. The pellet is collected by centrifugation at 25,000 g for 20 min and dissolved in a minimal volume of 100 m M Tris-HC1 buffer, pH 7.9. Step 4: Gel Filtration. The protein solution from Step 3 is applied to a column (2.6 × 35 cm) of Sephadex G-75 previously equilibrated with 20 m M Tris-HC1 buffer, pH 7.9. Methylamine dehydrogenase is eluted with equilibration buffer. Fractions containing activity are pooled. Step 5: DEAE- Toyopearl Chromatography. The enzyme solution from Step 4 is applied to a column (1.6 X 17 cm) of DEAE-Toyopead, equilibrated with 20 m M Tris-HC1 buffer, pH 7.9. The column is washed with 2 volumes of the same buffer (rate 0.8 ml/min). A portion of the methylamine dehydrogenase activity is eluted during washing. This fraction (Fraction 1) does not bind to the ion exchanger but is slightly retained on the column. Another portion of the activity is eluted with a linear gradient (200 ml) of 0 to 500 m M KC1 in starting buffer. Methylamine dehydrogenase is eluted in a single peak in the interval 25 to 50 m M of KC1. Fractions containing activity are pooled and adjusted with glycerol up to a s N. I. Govorukhina, L. V. Kletsova, Y. D. Tsygankov, Y. A. Trotsenko, and A. I. Netrusov, Mikrobiologija 56, 849 (1988).
[39]
MADH FROM M. flagellatum
249
concentration of 50% (Fraction 2). The glycerol solution of methylamine dehydrogenase is stored at - 2 0 °. A typical purification scheme is shown in Table I. Properties
Purity. The enzyme preparation of Fraction 2 is homogeneous according to gel filtration and electrophoresis data. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 9 the preparation exhibited two bands with Mr values of 43,000 and 16,000 after staining with Coomassie blue. Gel filtration on Sephacryl G-200 and fast protein liquid chromatography (FPLC) chromatography on a Superose 12 column (Pharmacia LKB) revealed one symmetrical peak with a n M r value 60,000 + 5,000. In both cases elution was carried out with 0.2 M NaC1 in 0.3 M Tris-HC1 buffer, pH 7.5. The enzyme from Fraction 1 is not homogeneous: analysis of 80 fig by SDS-PAGE showed a set of protein bands. Two of these bands correspond to bands of Fraction 2. They are major. Gel filtration under the same conditions reveals a peak with an Mr value 55,000 + 5,000. Isoelectric Focusing. Isoelectric focusing of methylamine dehydrogenase Fraction 2 showed two bands o f p I 7.40 and 7.80 after Coomassie blue staining. Isoelectric focusing of Fraction 1 showed one major band of pI 8.80. Substrate Specificity. Neither fraction oxidizes alanine, glycine, 1,4diaminobutane, lysine, 1-phenylethylamine, or dimethylamine. Activities with ethylamine comprise 20% for the Fraction 2 preparation and 43% for Fraction 1 in comparison with methylamine. Allylamine irreversibly inhibits both enzymes. Stability. Both enzyme fractions are stable at room temperature. Solutions in 100 m M Tris-HC1 buffer, pH 8.0, are stable for 1 month at 4°; in the same buffer with 50% glycerol they are stable for at least 4 months at - 2 0 °. Enzyme activities, under the assay conditions, increase linearly up to 75 ° (6-fold increase compare to 30°). Purified methylamine dehydrogenase does not change its activity after a 30-min incubation at 70 ° and loses 95% of activity after a 15-min incubation at 80 °. pH Optimum. The enzyme has optimum activity in both fractions at pH 7.5-8.0. Michaelis Constants. The apparent Km for the Fraction 2 enzyme is 1.4 × 10-5 M for methylamine (determined at 1.5 m M PMS concentration); for PMS it is 1.45 X 10-4 M (determined at 200 # M methylamine). 9 U. K. Laemmli, Nature (London) 227, 680 (1970).
250
METHYLOTROPHY
[40]
TABLE I PURIFICATIONOF METHYLAMINEDEHYDROGENASEFROM Methylobacillusflagellatum
Fraction Step 1. Cell-free extract Step 2. Heat treatment, 75", 20 min Step 3, 4. (NH4)2SO 4 fraction (0-70%) after Sephadex G-75 gel filtration Step 5. Pooled fraction 1 after washing DEAEToyopearl Step 5. Pooled fraction 2 after elution with linear gradient KC1 (0-0.5
Total Total Total volume (ml) protein (mg) activity (units)
Specific activity (units/mg protein)
Yield (%)
131 125
2096 1125
114 107
0.054 0.095
100 94
21
63
43
0.68
38
9
1.45
8
30
2.72
26
22
12
6.2
11
M) For the Fraction 1 enzyme the apparent Km for methylamine is 2 X 10-5 M (determined at 2.0 mM PMS); for PMS it is 2.10 X 10-4 M (determined at 200 pM methylamine). Molecular Weight and Subunit Structure. Methylamine dehydrogenase from Methylobacillusflagellatumis heterodimer (aft) (see above). Methylamine dehydrogenases from other bacteria have four subunits (a2f12).~-5
[40] Trimethylamine D e h y d r o g e n a s e from Bacterium W3A1 B y W I L L I A M S. M C I N T I R E (CH3)3NH + + H20 d- acceptor --, (CH3)2NH2+ -/- formaldehyde + reduced acceptor (CH3)2NH2 + "~-H20 d- acceptor ---, CH3NH3+ + formaldehyde + reduced acceptor
Trimethylamine dehydrogenase is the first enzyme involved in the assimilation of trimethylamine and dimethylamine by the gram-negative restricted facultative methylotrophs bacterium W3A 1t,2 and Hyphomicrobium X. 3 Trimethylamine dehydrogenase (EC 1.5.99.7) has been purified J. Colby and L. J. Zatman, Biochem. J. 148, 505 (1975). 20. Jenkins and D. Jones, J. Gen. Microbiol. 133, 453 (1987). 3 T. Urakami and K. Komagata, J. Gen. Appl. Microbiol. 33, 521 (1987); W. Harder and M. M. Atwood, Adv. Microb. Physiol. 17, 303 (1978). METHODS IN ENZYMOLOGY,VOL. 188
Copyright© 1990by AcademicPress,Inc. All fightsof reproductionin any formr'-~erved.
[39]
[39] M e t h y l a m i n e
Dehydrogenase
247
f r o m Methylobacillus
flagellatum By MICnA~.L Y. KmIUKmN, ANDREY Y. CI-nSTOSERDOV,and Y U R I D . TSYGANKOV
Methylamine + pyrroloquinolinc quinonc + H20 "-~ formaldehyde + reduced pyrroloquinolinc quinone
Methylamine dehydrogenase catalyzes the oxidation of methylamine to formaldehyde and ammonia in the presence of an artificial electron acceptor. The enzyme is induced during growth of the bacterium on mcthylamine. Methylamine dehydrogenases have been purified from various methylotrophic bacteria.t-5 Assay Method
Principle. The assay method involves the continuous measurement of the reduction of the artificial electron acceptor phenazine methosulfate (PMS)?2,6-Dichlorophenolindophenol (DCPIP) is used as a redox indicator. The molar extinction coefficient for DCPIP is taken from the work of Armstrong.7 Reagents
Potassium phosphate buffer, 200 raM, pH 7.5 Phenazine methosulfate, 10 m M 2,6-Dichlorophenolindophenol, 1 m M Methylamine hydrochloride, 200 m M Procedure. The assay mixture contains, in a final volume of 1 ml, potassium phosphate buffer, 0.5 ml; water, 0.19 ml; DCPIP, 0.1 ml; PMS, 0.1 ml; cell extract, 10 gl. The mixture is incubated for 5 min at 30 °. After preincubation 0.1 ml of methylamine hydrochloride is added, and the rate of decrease in absorbance is followed at 600 nm. 1 C. Anthony, "The Biochemistry of Methylotrophs." Academic Press, London, 1982. 2 M. Husain and V. Davidson, J. Bacteriol. 169, 1712 (1987). 3 W. C. Kenny and W. McIntir¢, Biochemistry 22, 3858 (1983). 4 G. W. Heywood, N. S. Janschke, P. J. Large, and J. M. Wallis, FEMS Microbiol. Lett. 15, 79 (1982). 5 F. M. D. VeUieux, M. D. J. Frank, M. B. A. Swarte, H. Groendijk, J. A. Duine, J. Drenth, and W. G. J. Hol, Eur. J. Biochem. 154, 383 (1986). 6 R. R. Eady and P. J. Large, Biochem. J. 106, 245 (1968). 7 j. Armstrong, Biochim. Biophys. Acta 86, 194 (1964).
METHODS IN ENZYMOLOGY, VOL. 188
English translation copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
248
METHYLOTROPHY
[39]
Units. One unit of methylamine dehydrogenase is defined as that amount of enzyme catalyzing reduction of 1/~mol of DCPIP per minute at 30 °. Specific activity is expressed as units per milligram of protein. Purification Procedure
Growth of Organism. The strain of Methylobacillus flagellatum s is grown at 42° in a fermentor in a mineral salts medium containing, per liter: Na2HPO4, 6 g; KH2PO4, 3 g; NaC1, 0.5 g; NH4C1, 1 g; CaC12, 0.11 g; MgSO4.7H20, 0.5 g; and methylamine, 3 g. After 72 hr of growth, the culture is harvested by~centrifugation at 3500 g for 60 min, and the pellet is rinsed with cooled 100 m M potassium phosphate buffer, pH 7.0. The cell paste is stored at - 2 0 °. Step 1: Preparation of Cell-Free Extract. Cell paste (30 g wet weight) is suspended in 150 ml of 100 m M Tris-HC1 buffer, pH 8.0. The suspension is disrupted by ultrasonication for 5 rain (10 times, 30 sec each) at 0 ° in an MSE-150 ultrasonic disintegrator and centrifuged at 25,000 g for 60 min, yielding the cell-free extract as supernatant. Step 2: Heat Treatment. The crude extract is heated at 75 ° for 20 min with slow mixing. This suspension is cooled to 5 ° and centrifuged at 25,000 g for 20 min. The supernatant is used further. Step 3: Ammonium Sulfate Fractionation. To the supernatant, solid (NH4)2SO4 is added with stirring up to 70% of saturation and kept at 4 ° for 1 hr. The pellet is collected by centrifugation at 25,000 g for 20 min and dissolved in a minimal volume of 100 m M Tris-HC1 buffer, pH 7.9. Step 4: Gel Filtration. The protein solution from Step 3 is applied to a column (2.6 × 35 cm) of Sephadex G-75 previously equilibrated with 20 m M Tris-HC1 buffer, pH 7.9. Methylamine dehydrogenase is eluted with equilibration buffer. Fractions containing activity are pooled. Step 5: DEAE- Toyopearl Chromatography. The enzyme solution from Step 4 is applied to a column (1.6 X 17 cm) of DEAE-Toyopead, equilibrated with 20 m M Tris-HC1 buffer, pH 7.9. The column is washed with 2 volumes of the same buffer (rate 0.8 ml/min). A portion of the methylamine dehydrogenase activity is eluted during washing. This fraction (Fraction 1) does not bind to the ion exchanger but is slightly retained on the column. Another portion of the activity is eluted with a linear gradient (200 ml) of 0 to 500 m M KC1 in starting buffer. Methylamine dehydrogenase is eluted in a single peak in the interval 25 to 50 m M of KC1. Fractions containing activity are pooled and adjusted with glycerol up to a s N. I. Govorukhina, L. V. Kletsova, Y. D. Tsygankov, Y. A. Trotsenko, and A. I. Netrusov, Mikrobiologija 56, 849 (1988).
[39]
MADH FROM M. flagellatum
249
concentration of 50% (Fraction 2). The glycerol solution of methylamine dehydrogenase is stored at - 2 0 °. A typical purification scheme is shown in Table I. Properties
Purity. The enzyme preparation of Fraction 2 is homogeneous according to gel filtration and electrophoresis data. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 9 the preparation exhibited two bands with Mr values of 43,000 and 16,000 after staining with Coomassie blue. Gel filtration on Sephacryl G-200 and fast protein liquid chromatography (FPLC) chromatography on a Superose 12 column (Pharmacia LKB) revealed one symmetrical peak with a n M r value 60,000 + 5,000. In both cases elution was carried out with 0.2 M NaC1 in 0.3 M Tris-HC1 buffer, pH 7.5. The enzyme from Fraction 1 is not homogeneous: analysis of 80 fig by SDS-PAGE showed a set of protein bands. Two of these bands correspond to bands of Fraction 2. They are major. Gel filtration under the same conditions reveals a peak with an Mr value 55,000 + 5,000. Isoelectric Focusing. Isoelectric focusing of methylamine dehydrogenase Fraction 2 showed two bands o f p I 7.40 and 7.80 after Coomassie blue staining. Isoelectric focusing of Fraction 1 showed one major band of pI 8.80. Substrate Specificity. Neither fraction oxidizes alanine, glycine, 1,4diaminobutane, lysine, 1-phenylethylamine, or dimethylamine. Activities with ethylamine comprise 20% for the Fraction 2 preparation and 43% for Fraction 1 in comparison with methylamine. Allylamine irreversibly inhibits both enzymes. Stability. Both enzyme fractions are stable at room temperature. Solutions in 100 m M Tris-HC1 buffer, pH 8.0, are stable for 1 month at 4°; in the same buffer with 50% glycerol they are stable for at least 4 months at - 2 0 °. Enzyme activities, under the assay conditions, increase linearly up to 75 ° (6-fold increase compare to 30°). Purified methylamine dehydrogenase does not change its activity after a 30-min incubation at 70 ° and loses 95% of activity after a 15-min incubation at 80 °. pH Optimum. The enzyme has optimum activity in both fractions at pH 7.5-8.0. Michaelis Constants. The apparent Km for the Fraction 2 enzyme is 1.4 × 10-5 M for methylamine (determined at 1.5 m M PMS concentration); for PMS it is 1.45 X 10-4 M (determined at 200 # M methylamine). 9 U. K. Laemmli, Nature (London) 227, 680 (1970).
250
METHYLOTROPHY
[40]
TABLE I PURIFICATIONOF METHYLAMINEDEHYDROGENASEFROM Methylobacillusflagellatum
Fraction Step 1. Cell-free extract Step 2. Heat treatment, 75", 20 min Step 3, 4. (NH4)2SO 4 fraction (0-70%) after Sephadex G-75 gel filtration Step 5. Pooled fraction 1 after washing DEAEToyopearl Step 5. Pooled fraction 2 after elution with linear gradient KC1 (0-0.5
Total Total Total volume (ml) protein (mg) activity (units)
Specific activity (units/mg protein)
Yield (%)
131 125
2096 1125
114 107
0.054 0.095
100 94
21
63
43
0.68
38
9
1.45
8
30
2.72
26
22
12
6.2
11
M) For the Fraction 1 enzyme the apparent Km for methylamine is 2 X 10-5 M (determined at 2.0 mM PMS); for PMS it is 2.10 X 10-4 M (determined at 200 pM methylamine). Molecular Weight and Subunit Structure. Methylamine dehydrogenase from Methylobacillusflagellatumis heterodimer (aft) (see above). Methylamine dehydrogenases from other bacteria have four subunits (a2f12).~-5
[40] Trimethylamine D e h y d r o g e n a s e from Bacterium W3A1 B y W I L L I A M S. M C I N T I R E (CH3)3NH + + H20 d- acceptor --, (CH3)2NH2+ -/- formaldehyde + reduced acceptor (CH3)2NH2 + "~-H20 d- acceptor ---, CH3NH3+ + formaldehyde + reduced acceptor
Trimethylamine dehydrogenase is the first enzyme involved in the assimilation of trimethylamine and dimethylamine by the gram-negative restricted facultative methylotrophs bacterium W3A 1t,2 and Hyphomicrobium X. 3 Trimethylamine dehydrogenase (EC 1.5.99.7) has been purified J. Colby and L. J. Zatman, Biochem. J. 148, 505 (1975). 20. Jenkins and D. Jones, J. Gen. Microbiol. 133, 453 (1987). 3 T. Urakami and K. Komagata, J. Gen. Appl. Microbiol. 33, 521 (1987); W. Harder and M. M. Atwood, Adv. Microb. Physiol. 17, 303 (1978). METHODS IN ENZYMOLOGY,VOL. 188
Copyright© 1990by AcademicPress,Inc. All fightsof reproductionin any formr'-~erved.