- Email: [email protected]
[17] Gentisate 1,2-dioxygenase from Pseudomonas acidovorans
[17]
101
GENTISATE 1,2-DIOXYGENASE
oxide, 6-hydroxynicotinic acid N-oxide, and 4-nitrocatechol, are tight binding inhibitors. Spectroscopic Properties. The enzyme as isolated exhibits no optical spectrum in the visible range. Preparations exhibit a weak electron paramagnetic resonance (EPR) spectrum from contaminating Mn 2+ often found associated with enzymes isolated from Bacillus. This metal is removed without activity loss by the same procedure which removes adventitious iron. The active site Fe(II) reversibly binds nitric oxide to produce an EPR-active complex that exhibits a spectrum characteristic of an S = 3/2 system. The spectrum is altered by substrate and inhibitor binding, and direct coordination of substrate to the iron has been demonstrated in this complex through the use of specific isotopic labeling. 3,7 Acknowledgment This work was supported by a grant from the National Institutes of Health (GM 24689). 7 S. A. Wolgel and J. D. Lipscomb, Fed. Proc., Fed. Am. Soc. Exp. Biol. 45, 1521 (1986).
[ 17] G e n t i s a t e
from Pseudomonas
1,2-Dioxygenase
acidovorans By MARK R. HARPEL a n d JOHN D. LIPSCOMB
HO~
I
~
COOH
COOH
OH
02
Gentisic Acid
~-
O COOH
Maleylpyruvic Acid
Gentisate 1,2-dioxygenase ~ catalyzes the cleavage of gentisate and related structures in the environment. Gentisate serves as the focal point in the biodegradation of a large number of simple and complex aromatic
I EC 1.13.11.4, gentisate: oxygen oxidoreductase METHODSIN ENZYMOLOGY,VOL.188
Copyright© 1990byAcademicPress,Inc. Allrightsofreproductionin anyformreserved.
102
HYDROCARBONS AND RELATED COMPOUNDS
[ 17]
compounds. 2,3 Consequently, the gentisate pathway is distributed throughout the microbial world.4-8 A purification of gentisate 1,2-dioxygenase from Moraxella osloensis has been published? The preparation from Pseudomonas acidovorans described here exhibits approximately a 40-fold higher specific activity and does not require postpurification activation.m° Assay Method
Principle. Gentisate 1,2-dioxygenase activity can be conveniently measured by monitoring the time course of either reactant (oxygen) depletion or product (maleylpyruvate) formation. The former technique involves polarographic measurement of enzyme-stimulated oxygen consumption in the presence of gentisate. The latter technique involves optical measurement of the increase of absorbance at 330 nm arising from the formation of maleylpyruvate. The polarographic assay has been found to be more sensitive and better suited for kinetic studies. A description of the optical assay can be found elsewhere.H Reagents MOPS ~2buffer, 50 raM, containing 10% glycerol, adjusted to pH 7.4 with NaOH Gentisic acid, charcoal-treated and recrystallized from hot water Gentisate stock solution, 0.1 M, prepared anaerobically using recrystallized gentisic acid neutralized with 1 equivalent of NaOH and stored under argon in a sealed septum vial Procedure. Gentisate 1,2-dioxygenase activity is measured by the calibrated oxygen uptake assay using a Clark-type oxygen electrode as described elsewhere in this volume. 13Gentisate is added to the assay chamber to a final concentration of 1.25 mM from the 0.1 M stock solution. Stan2 p. j. Chapman, in "Degradation of Synthetic Organic Molecules in the Biosphere," (I.C. Gunsalus, ed.), p. 17. National Academy of Sciences, Washington, D.C., 1972. 3 R. C. Bayly and M. G. Barbour, in "Microbial Degradation of Organic Molecules" (D. T. Gibson, ed.), p. 253. Dekker, New York, 1978. 4 L. Lack, Biochim. Biophys. Acta 34, 117 (1959). 5 D. J. Hopper, P. J. Chapman, and S. Dagley, Biochem. J. 122, 29 (1971). 6 R. L. Crawford, J. Baeteriol. 127, 204 (1976). 7 j. N. Ladd, Nature (London) 194, 1099 (1962). s R. C. Crawford, J. BacterioL 167, 818 (1986). 9 R. C. Crawford, S. W. Hutton, and P. J. Chapman, J. Bacteriol. 121, 794 (1975). ~oM. R. Harpel and J. D. Lipscomb, J. Biol. Chem. 265, 6301 (1990). H M. L. Wheelis, N. J. Palleroni, and R. Y. Stanier, Arch. Mikrobiol. 59, 302 (1967). t2 MOPS, 3-(N-Morpholino)propanesulfonic acid. ~3j. W. Whittaker, A. M. Orville, and J. D. Lipscomb, this volume [14].
[17]
GENTISATE 1,2-DIOXYGENASE
103
dard dioxygenase assays are carried out at 23* and atmospheric oxygen concentrations. The assay reaction is initiated by addition of enzyme. Gentisate and its analogs are prone to autoxidation resulting in a small background level of oxygen consumption that must be accounted for. Units and Enzyme Concentration. One unit is defined as the consumption of 1/zmol of 02 per minute as determined above. This corresponds to turnover of 1 /zmol of gentisate per minute. Specific activity is given as units per milligram of protein. Enzyme concentration is determined colorimetrically ~4 versus bovine serum albumin. Purification P r o c e d u r e
Bacterial Growth. Pseudomonas acidovorans (ATCC 17438) is grown under inducing conditions for gentisate 1,2-dioxygenase and the immediate enzymes of the gentisate pathway. The bacterium is cultured in the minimal medium of Stanier ~5 supplemented with 0.2 g/liter yeast extract and 3 g/liter neutralized 3-hydroxybenzoic acid (free acid, Eastman Kodak Co., Rochester, NY). Per liter, the minimal medium contains 2.78 g Na2HPO4, 2.78 g KH2PO4, 1 g NH4(SO)4 , and 20 ml Hutner's vitaminfree concentrated mineral base. ~6 The Hutner's base is autoclaved separately from the rest of the medium, then aseptically added to it. The 3-hydroxybenzoic acid is neutralized by aseptically adding 3.6 ml of 6 N NaOH per liter of the autoclaved medium. The bacteria are maintained on agar containing the same medium. Large-scale growth experiments are carried out in 18-liter carboys conraining 15 liters of medium. A 100 ml liquid culture is inoculated from the agar maintenance culture and grown overnight at 30 ° with controlled shaking at 250 rpm in an environmental incubator. Then 1000 ml of liquid medium in a 2000-ml shake flask is inoculated with 10 ml of the overnight culture. This is grown under the same conditions. When the optical density of the culture reaches 0.15-0.2 at 650nm, indicating mid-late log phase of growth, the entire culture is used to inoculate one carboy of medium. This culture is incubated at room temperature with forced aeration from filter-sterilized air at a flow rate of approximately 95 liter/min. An antifoaming agent (2 ml Antifoam A Emulsion, Sigma, St. Louis, MO) is added to the carboy at this time, and as necessary through14M. Bradford, Anal. Biochem. 72, 248 (1976). 13R. Y. Stanier, N. J. Palleroni, and M. Doudoroff, J. Gen. Microbiol. 43, 236 (1966). 16G. Cohen-Bazire, W. R. Sistrom, and R. Y. Stanier, J. Cell. Comp. Physiol. 49, 25 (1957); R. M. Smibert and N. R. Krieg, in "Manual of Methods for General Bacteriology," (P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B. Phillips, eds.), p. 434. American Society for Microbiology, Washington, D.C., 1981.
104
HYDROCARBONS AND RELATED COMPOUNDS
[ 17]
out the growth to avoid excessive foaming of the culture. The concentration of 3-hydroxybenzoate is monitored during the growth by measuring the absorbance of centrifuged samples at 288 nm. When the growth substrate is 70- 80% depleted (after - 24 hr), the culture is supplemented with 45 g of neutralized 3-hydroxybenzoate. When growth substrate becomes depleted for the second time ( - 12 hr additional time) the culture is harvested with a Sharpies continuous-flow centrifuge. Growth yields are typically 10 g/liter wet weight. Cell paste is stored at - 20 °. Purification of gentisate 1,2-dioxygenase is performed at 4 °. Buffers are 50 m M MOPS, 10% glycerol (EM Science, Cherry Hill, NJ, ACS grade), pH 7.4, containing freshly prepared 100/tM ferrous ammonium sulfate and 2 m M L-cysteine. The reduced iron is essential for retaining full gentisate 1,2-dioxygenase activity throughout the purification. Table I shows a typical purification scheme. Step 1: Cell-Free Extract. Cell paste ( 150 g) is resuspended in 300 ml of buffer. Approximately 1 mg each of ribonuclease and deoxydbonuclease are added. The cells are disrupted by sonication (Branson Sonifier, 14 kHz output) for 25 min, maintaining the solution temperature at 4 - 10 ° with a 2-propranol-dry ice bath. The suspension is then centrifuged for 30 min at 8875 g. Step 2: Heat Treatment. Gentisate 1,2-dioxygenase is stable in the centrifuged cell-free extract to heating to 60*. The decanted supernatant from Step 1 is heated slowly with constant stirring in a 65 ° bath. When the protein solution reaches 60 °, it is removed from the bath and immediately cooled to about 15 ° in a dry ice-2-propanol slurry. The denatured protein is removed by centrifugation at 8875 g for 45 rain. This step irreversibly denatures approximately 80% of the total protein from the cell-free extract, with an 80% recovery of gentisate 1,2-dioxygenase activity in the supernarant. Step 3: Ammonium Sulfate Fractionation. The heat denaturation supernatant is further fractionated by ammonium sulfate precipitation. The pH of the heat step supematant is first readjusted to pH 7.4 by dropwise addition of 6 N NaOH. The solution is then brought to 33% saturation (196 g/liter) with ammonium sulfate (Ultrapure, Schwarz/Mann Biotech) by the addition of ammonium sulfate powder over a period of 30 min with constant stirring. NaOH (6 N) is added dropwise as necessary to maintain the pH at 7.4. The suspension is equilibrated for an additional 30 min, then centrifuged for 30 min (8875 g). The supernatant contains the gentisate 1,2-dioxygenase activity. This is decanted and brought to 40% ammonium sulfate saturation (additional 43 g/liter) in the same manner as described above, then centrifuged. The supernatant is free of gentisate 1,2-dioxygenase activity and is discarded. Step 4: Fast-Flow DEAE-Sepharose CL-6B Chromatography. The 40%
[17]
105
GENTISATE 1,2-DIOXYGENASE TABLE I PURIFICATIONOF GENTISATE 1,2-DIOXYGENASEFROM Pseudomonas acidovorans
Step Cell-free extract a 60* Supernatant 33-40% (NH4)2SO 4 precipitation Fast-flow DEAESepharose CL-6B Phenyl-Sepharose CL-4B (PM30 concentration)
Volume (ml) 375 257 242
Protein (mg) 4460 810 200
Units (umol O~Jmin)
RecoveD (%)
Specific activity (units/rag)
(-fold) Purification
35,020 28,140 27,190
100 80 78
7.9 34.8 136
1 4.4 17.2
182
44.0
20,120
58
461
58.4
19
22.0
10,670
31
484
61.3
a From 146 g wet weight cell paste.
ammonium sulfate pellet is resuspended in buffer to a conductivity of no more than 3 mS (Radiometer CDM83 Conductivity Meter, 3.16 mS/cm cell constant) and loaded onto a column of Fast-Flow DEAE-Sepharose CL-6B (Pharmacia, 4.5 × 14 cm) that had been previously equilibrated in buffer containing Fe 2+ and cysteine as described above. The column is washed with 200 ml buffer, then 500 ml of 0.1 M NaCI in buffer. The gentisate 1,2-dioxygenase activity is eluted with a gradient (1.8 liter) from 0.1 to 0.22 M NaC1 in buffer. Fractions containing activities 30% or greater of the peak fraction are pooled. The flow rate is maintained at 31.5 cm/hr. Step 5: Phenyl-Sepharose CL-4B Chromatography. The pooled fractions from Step 4 are brought to 0.5 M in ammonium sulfate and loaded onto a column of Phenyl-Sepharose CL-4B (Pharmacia, 2.5 × 8 cm) that had been preequilibrated in buffer containing 1 M ammonium sulfate. The column is washed with 100 ml of 0.4 M ammonium sulfate in buffer, and then a reverse salt gradient (300 ml) is run from 0.4 to 0 M ammonium sulfate in buffer. The gentisate 1,2-dioxygenase activity begins to elute approximately halfway through this gradient, and fractions with activity of 30% or greater of the peak fraction are pooled. The pool is concentrated by ultrafiltration under N2 (Amicon PM30 membrane), with several cycles of resuspension in buffer containing no iron or cysteine. Any remaining adventitiously bound iron can be removed by anaerobic treatment with the Fea+-specific chelator Tiron (4,5-dihydroxy-l,3-benzenedisulfonic acid). 17 The purified and concentrated gentisate 1,2-dioxygenase is stored at - 80 °
17 D. M. Arciero, J. D. Lipscomb, B. H. Huynh, T. A. Kent, and E. Miinck, J. Biol. Chem. 258, 14981 (1983).
106
HYDROCARBONS AND RELATED COMPOUNDS
[ 17]
with minimal loss of activity, retaining at least 85% of full activity for 1 year. Properties
Physical Properties. Amino-terminal sequencing of purified gentisate 1,2-dioxygenase shows only a single high-yield polypeptide. A single band of protein is also seen in native gel electrophoresis. Denaturing electrophoresis (SDS-PAGE) reveals a major band at 39,800 molecular weight with two diffuse bands at slightly greater relative mobility, which related studies suggest arise from a microheterogeneity within the single subunit type of this enzyme. The holoenzyme molecular mass determined by gel-permeation chromatography on BioGel A0.5 M (Bio-Rad, Richmond, CA) is 164 kDa. This indicates an cq subunit structure. Isoelectric focusing of the protein reveals a pI of 4.55-4.8. Inductively coupled plasma emission analysis and colorimetric assays indicate that one equivalent of iron per subunit is present in fully active protein. The enzyme as isolated is essentially electron paramagnetic resonance (EPR) silent. However, the anaerobic complex with nitric oxide (NO) is EPR active (S--- 3/2). Quantitation of this signal shows that a 1 : 1 complex of NO to the iron is formed. The appearance of this type of signal is diagnostic of Fe 2+ in the enzyme as isolated) 7,18The signal is perturbed by substrate binding. Kinetic Properties and Stability. The enzyme exhibits a broad maxim u m of activity from pH 7 to 9. It is most stable near pH 7.4, showing a sharp drop in stability below pH 6 or above pH 8. It is relatively unstable in dilute solutions not containing the stabilizers glycerol, Fe 2+, and cysteine. At atmospheric oxygen tension ( - 250 p.M), the apparent Michaelis constant for gentisate equals 80 #M. The turnover number under these conditions is 19,300 min-' (per active site). The K,,~,p for 02 is determined to be 55 pM. A K,,.~a, of 57/tM for gentisate at saturating [02] is derived from intercept replots with 02 as the fixed changing substrate. The estimated turnover number at saturating oxygen concentration is approximately 24,000 rain- t (per active site). Under turnover conditions, gentisate 1,2-dioxygenase is rapidly inactivated at oxygen concentrations above 800 aM. Substrate Specificity and Inhibition. Gentisate 1,2-dioxygenase from P. acidovorans catalyzes the turnover of a range of alkyl- and halo-substituted gentisates. Somewhat reduced rates are observed with gentisate containing substitutions (methyl, ethyl, 2-propyl, bromo, fluoro) at the ring C-3 position. Analogs of gentisate with similar substitutions in either the C-4 or in t8 j. C. Salerno and J. N. Siedow, Biochim. Biophys. Acta 579, 246 (1979).
[18]
SYNTHESIS OF DIHYDROXY AROMATIC COMPOUNDS
107
both the C-3 and C-4 positions are also turned over, but at dramatically reduced rates (< 10% that ofgentisate). All three main functional groups of gentisate appear to be required for efficient turnover, in that little or no turnover is observed with compounds in which any one of these groups is substituted with another functionality. Additionally, monosubstituted benzoates such as salicylate, 3-hydroxybenzoate, and thiosalicylate are not turned over but are relatively good inhibitors of the enzyme. Gentisate 1,2-dioxygenase is inactivated in a time- and concentration-dependent manner by oxidants such as H202 and K3Fe(CN)6. Partial reactivation is effected by ascorbate. Acknowledgment This work was supported by a grant from the National Institutes of Health (GM 24689).
[18] S y n t h e s i s o f 170- o r 1 s O - E n r i c h e d D i h y d r o x y Aromatic Compounds B y A L L E N M . ORVILLE, M A R K R . H A R P E L , a n d JOHN D . LIPSCOMB
Specific isotopic labeling of the oxygen atoms of phenolic and catecholic compounds is of use in spectroscopic and kinetic investigations of a variety of enzymes that bind these molecules as substrates or inhibitors.t.2 A convenient and versatile synthetic procedure is depicted in Scheme 1. The position of incorporation of the isotopically enriched oxygen atom is determined by the source of the enriched oxygen (H20 or 02) and the position of substituents in the starting aromatic amine. This methodology is illustrated here with the synthesis of isotopically enriched homoprotocatechuate (3,4-dihydroxyphenylacetate, HPCA a) as an example. This procedure has appeared in a previous report. 2 These techniques have been shown to be applicable to a wide variety of phenolic and catecholic aromatic compounds (Table I).
1D. M. Arciero and J. D. Lipscomb, J. Biol. Chem. 261, 2170 (1986). 2 A. M. Orville and J. D. Lipscomb, J. Biol. Chem. 264, 8791 (1989). 3 Abbreviations used are given in Table I.
METHODS IN ENZYMOLOGY,VOL. 188
Copyright© 1990by AcademicPress,Inc. Allfightsofreproductionin any formreserved.
101
GENTISATE 1,2-DIOXYGENASE
oxide, 6-hydroxynicotinic acid N-oxide, and 4-nitrocatechol, are tight binding inhibitors. Spectroscopic Properties. The enzyme as isolated exhibits no optical spectrum in the visible range. Preparations exhibit a weak electron paramagnetic resonance (EPR) spectrum from contaminating Mn 2+ often found associated with enzymes isolated from Bacillus. This metal is removed without activity loss by the same procedure which removes adventitious iron. The active site Fe(II) reversibly binds nitric oxide to produce an EPR-active complex that exhibits a spectrum characteristic of an S = 3/2 system. The spectrum is altered by substrate and inhibitor binding, and direct coordination of substrate to the iron has been demonstrated in this complex through the use of specific isotopic labeling. 3,7 Acknowledgment This work was supported by a grant from the National Institutes of Health (GM 24689). 7 S. A. Wolgel and J. D. Lipscomb, Fed. Proc., Fed. Am. Soc. Exp. Biol. 45, 1521 (1986).
[ 17] G e n t i s a t e
from Pseudomonas
1,2-Dioxygenase
acidovorans By MARK R. HARPEL a n d JOHN D. LIPSCOMB
HO~
I
~
COOH
COOH
OH
02
Gentisic Acid
~-
O COOH
Maleylpyruvic Acid
Gentisate 1,2-dioxygenase ~ catalyzes the cleavage of gentisate and related structures in the environment. Gentisate serves as the focal point in the biodegradation of a large number of simple and complex aromatic
I EC 1.13.11.4, gentisate: oxygen oxidoreductase METHODSIN ENZYMOLOGY,VOL.188
Copyright© 1990byAcademicPress,Inc. Allrightsofreproductionin anyformreserved.
102
HYDROCARBONS AND RELATED COMPOUNDS
[ 17]
compounds. 2,3 Consequently, the gentisate pathway is distributed throughout the microbial world.4-8 A purification of gentisate 1,2-dioxygenase from Moraxella osloensis has been published? The preparation from Pseudomonas acidovorans described here exhibits approximately a 40-fold higher specific activity and does not require postpurification activation.m° Assay Method
Principle. Gentisate 1,2-dioxygenase activity can be conveniently measured by monitoring the time course of either reactant (oxygen) depletion or product (maleylpyruvate) formation. The former technique involves polarographic measurement of enzyme-stimulated oxygen consumption in the presence of gentisate. The latter technique involves optical measurement of the increase of absorbance at 330 nm arising from the formation of maleylpyruvate. The polarographic assay has been found to be more sensitive and better suited for kinetic studies. A description of the optical assay can be found elsewhere.H Reagents MOPS ~2buffer, 50 raM, containing 10% glycerol, adjusted to pH 7.4 with NaOH Gentisic acid, charcoal-treated and recrystallized from hot water Gentisate stock solution, 0.1 M, prepared anaerobically using recrystallized gentisic acid neutralized with 1 equivalent of NaOH and stored under argon in a sealed septum vial Procedure. Gentisate 1,2-dioxygenase activity is measured by the calibrated oxygen uptake assay using a Clark-type oxygen electrode as described elsewhere in this volume. 13Gentisate is added to the assay chamber to a final concentration of 1.25 mM from the 0.1 M stock solution. Stan2 p. j. Chapman, in "Degradation of Synthetic Organic Molecules in the Biosphere," (I.C. Gunsalus, ed.), p. 17. National Academy of Sciences, Washington, D.C., 1972. 3 R. C. Bayly and M. G. Barbour, in "Microbial Degradation of Organic Molecules" (D. T. Gibson, ed.), p. 253. Dekker, New York, 1978. 4 L. Lack, Biochim. Biophys. Acta 34, 117 (1959). 5 D. J. Hopper, P. J. Chapman, and S. Dagley, Biochem. J. 122, 29 (1971). 6 R. L. Crawford, J. Baeteriol. 127, 204 (1976). 7 j. N. Ladd, Nature (London) 194, 1099 (1962). s R. C. Crawford, J. BacterioL 167, 818 (1986). 9 R. C. Crawford, S. W. Hutton, and P. J. Chapman, J. Bacteriol. 121, 794 (1975). ~oM. R. Harpel and J. D. Lipscomb, J. Biol. Chem. 265, 6301 (1990). H M. L. Wheelis, N. J. Palleroni, and R. Y. Stanier, Arch. Mikrobiol. 59, 302 (1967). t2 MOPS, 3-(N-Morpholino)propanesulfonic acid. ~3j. W. Whittaker, A. M. Orville, and J. D. Lipscomb, this volume [14].
[17]
GENTISATE 1,2-DIOXYGENASE
103
dard dioxygenase assays are carried out at 23* and atmospheric oxygen concentrations. The assay reaction is initiated by addition of enzyme. Gentisate and its analogs are prone to autoxidation resulting in a small background level of oxygen consumption that must be accounted for. Units and Enzyme Concentration. One unit is defined as the consumption of 1/zmol of 02 per minute as determined above. This corresponds to turnover of 1 /zmol of gentisate per minute. Specific activity is given as units per milligram of protein. Enzyme concentration is determined colorimetrically ~4 versus bovine serum albumin. Purification P r o c e d u r e
Bacterial Growth. Pseudomonas acidovorans (ATCC 17438) is grown under inducing conditions for gentisate 1,2-dioxygenase and the immediate enzymes of the gentisate pathway. The bacterium is cultured in the minimal medium of Stanier ~5 supplemented with 0.2 g/liter yeast extract and 3 g/liter neutralized 3-hydroxybenzoic acid (free acid, Eastman Kodak Co., Rochester, NY). Per liter, the minimal medium contains 2.78 g Na2HPO4, 2.78 g KH2PO4, 1 g NH4(SO)4 , and 20 ml Hutner's vitaminfree concentrated mineral base. ~6 The Hutner's base is autoclaved separately from the rest of the medium, then aseptically added to it. The 3-hydroxybenzoic acid is neutralized by aseptically adding 3.6 ml of 6 N NaOH per liter of the autoclaved medium. The bacteria are maintained on agar containing the same medium. Large-scale growth experiments are carried out in 18-liter carboys conraining 15 liters of medium. A 100 ml liquid culture is inoculated from the agar maintenance culture and grown overnight at 30 ° with controlled shaking at 250 rpm in an environmental incubator. Then 1000 ml of liquid medium in a 2000-ml shake flask is inoculated with 10 ml of the overnight culture. This is grown under the same conditions. When the optical density of the culture reaches 0.15-0.2 at 650nm, indicating mid-late log phase of growth, the entire culture is used to inoculate one carboy of medium. This culture is incubated at room temperature with forced aeration from filter-sterilized air at a flow rate of approximately 95 liter/min. An antifoaming agent (2 ml Antifoam A Emulsion, Sigma, St. Louis, MO) is added to the carboy at this time, and as necessary through14M. Bradford, Anal. Biochem. 72, 248 (1976). 13R. Y. Stanier, N. J. Palleroni, and M. Doudoroff, J. Gen. Microbiol. 43, 236 (1966). 16G. Cohen-Bazire, W. R. Sistrom, and R. Y. Stanier, J. Cell. Comp. Physiol. 49, 25 (1957); R. M. Smibert and N. R. Krieg, in "Manual of Methods for General Bacteriology," (P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B. Phillips, eds.), p. 434. American Society for Microbiology, Washington, D.C., 1981.
104
HYDROCARBONS AND RELATED COMPOUNDS
[ 17]
out the growth to avoid excessive foaming of the culture. The concentration of 3-hydroxybenzoate is monitored during the growth by measuring the absorbance of centrifuged samples at 288 nm. When the growth substrate is 70- 80% depleted (after - 24 hr), the culture is supplemented with 45 g of neutralized 3-hydroxybenzoate. When growth substrate becomes depleted for the second time ( - 12 hr additional time) the culture is harvested with a Sharpies continuous-flow centrifuge. Growth yields are typically 10 g/liter wet weight. Cell paste is stored at - 20 °. Purification of gentisate 1,2-dioxygenase is performed at 4 °. Buffers are 50 m M MOPS, 10% glycerol (EM Science, Cherry Hill, NJ, ACS grade), pH 7.4, containing freshly prepared 100/tM ferrous ammonium sulfate and 2 m M L-cysteine. The reduced iron is essential for retaining full gentisate 1,2-dioxygenase activity throughout the purification. Table I shows a typical purification scheme. Step 1: Cell-Free Extract. Cell paste ( 150 g) is resuspended in 300 ml of buffer. Approximately 1 mg each of ribonuclease and deoxydbonuclease are added. The cells are disrupted by sonication (Branson Sonifier, 14 kHz output) for 25 min, maintaining the solution temperature at 4 - 10 ° with a 2-propranol-dry ice bath. The suspension is then centrifuged for 30 min at 8875 g. Step 2: Heat Treatment. Gentisate 1,2-dioxygenase is stable in the centrifuged cell-free extract to heating to 60*. The decanted supernatant from Step 1 is heated slowly with constant stirring in a 65 ° bath. When the protein solution reaches 60 °, it is removed from the bath and immediately cooled to about 15 ° in a dry ice-2-propanol slurry. The denatured protein is removed by centrifugation at 8875 g for 45 rain. This step irreversibly denatures approximately 80% of the total protein from the cell-free extract, with an 80% recovery of gentisate 1,2-dioxygenase activity in the supernarant. Step 3: Ammonium Sulfate Fractionation. The heat denaturation supernatant is further fractionated by ammonium sulfate precipitation. The pH of the heat step supematant is first readjusted to pH 7.4 by dropwise addition of 6 N NaOH. The solution is then brought to 33% saturation (196 g/liter) with ammonium sulfate (Ultrapure, Schwarz/Mann Biotech) by the addition of ammonium sulfate powder over a period of 30 min with constant stirring. NaOH (6 N) is added dropwise as necessary to maintain the pH at 7.4. The suspension is equilibrated for an additional 30 min, then centrifuged for 30 min (8875 g). The supernatant contains the gentisate 1,2-dioxygenase activity. This is decanted and brought to 40% ammonium sulfate saturation (additional 43 g/liter) in the same manner as described above, then centrifuged. The supernatant is free of gentisate 1,2-dioxygenase activity and is discarded. Step 4: Fast-Flow DEAE-Sepharose CL-6B Chromatography. The 40%
[17]
105
GENTISATE 1,2-DIOXYGENASE TABLE I PURIFICATIONOF GENTISATE 1,2-DIOXYGENASEFROM Pseudomonas acidovorans
Step Cell-free extract a 60* Supernatant 33-40% (NH4)2SO 4 precipitation Fast-flow DEAESepharose CL-6B Phenyl-Sepharose CL-4B (PM30 concentration)
Volume (ml) 375 257 242
Protein (mg) 4460 810 200
Units (umol O~Jmin)
RecoveD (%)
Specific activity (units/rag)
(-fold) Purification
35,020 28,140 27,190
100 80 78
7.9 34.8 136
1 4.4 17.2
182
44.0
20,120
58
461
58.4
19
22.0
10,670
31
484
61.3
a From 146 g wet weight cell paste.
ammonium sulfate pellet is resuspended in buffer to a conductivity of no more than 3 mS (Radiometer CDM83 Conductivity Meter, 3.16 mS/cm cell constant) and loaded onto a column of Fast-Flow DEAE-Sepharose CL-6B (Pharmacia, 4.5 × 14 cm) that had been previously equilibrated in buffer containing Fe 2+ and cysteine as described above. The column is washed with 200 ml buffer, then 500 ml of 0.1 M NaCI in buffer. The gentisate 1,2-dioxygenase activity is eluted with a gradient (1.8 liter) from 0.1 to 0.22 M NaC1 in buffer. Fractions containing activities 30% or greater of the peak fraction are pooled. The flow rate is maintained at 31.5 cm/hr. Step 5: Phenyl-Sepharose CL-4B Chromatography. The pooled fractions from Step 4 are brought to 0.5 M in ammonium sulfate and loaded onto a column of Phenyl-Sepharose CL-4B (Pharmacia, 2.5 × 8 cm) that had been preequilibrated in buffer containing 1 M ammonium sulfate. The column is washed with 100 ml of 0.4 M ammonium sulfate in buffer, and then a reverse salt gradient (300 ml) is run from 0.4 to 0 M ammonium sulfate in buffer. The gentisate 1,2-dioxygenase activity begins to elute approximately halfway through this gradient, and fractions with activity of 30% or greater of the peak fraction are pooled. The pool is concentrated by ultrafiltration under N2 (Amicon PM30 membrane), with several cycles of resuspension in buffer containing no iron or cysteine. Any remaining adventitiously bound iron can be removed by anaerobic treatment with the Fea+-specific chelator Tiron (4,5-dihydroxy-l,3-benzenedisulfonic acid). 17 The purified and concentrated gentisate 1,2-dioxygenase is stored at - 80 °
17 D. M. Arciero, J. D. Lipscomb, B. H. Huynh, T. A. Kent, and E. Miinck, J. Biol. Chem. 258, 14981 (1983).
106
HYDROCARBONS AND RELATED COMPOUNDS
[ 17]
with minimal loss of activity, retaining at least 85% of full activity for 1 year. Properties
Physical Properties. Amino-terminal sequencing of purified gentisate 1,2-dioxygenase shows only a single high-yield polypeptide. A single band of protein is also seen in native gel electrophoresis. Denaturing electrophoresis (SDS-PAGE) reveals a major band at 39,800 molecular weight with two diffuse bands at slightly greater relative mobility, which related studies suggest arise from a microheterogeneity within the single subunit type of this enzyme. The holoenzyme molecular mass determined by gel-permeation chromatography on BioGel A0.5 M (Bio-Rad, Richmond, CA) is 164 kDa. This indicates an cq subunit structure. Isoelectric focusing of the protein reveals a pI of 4.55-4.8. Inductively coupled plasma emission analysis and colorimetric assays indicate that one equivalent of iron per subunit is present in fully active protein. The enzyme as isolated is essentially electron paramagnetic resonance (EPR) silent. However, the anaerobic complex with nitric oxide (NO) is EPR active (S--- 3/2). Quantitation of this signal shows that a 1 : 1 complex of NO to the iron is formed. The appearance of this type of signal is diagnostic of Fe 2+ in the enzyme as isolated) 7,18The signal is perturbed by substrate binding. Kinetic Properties and Stability. The enzyme exhibits a broad maxim u m of activity from pH 7 to 9. It is most stable near pH 7.4, showing a sharp drop in stability below pH 6 or above pH 8. It is relatively unstable in dilute solutions not containing the stabilizers glycerol, Fe 2+, and cysteine. At atmospheric oxygen tension ( - 250 p.M), the apparent Michaelis constant for gentisate equals 80 #M. The turnover number under these conditions is 19,300 min-' (per active site). The K,,~,p for 02 is determined to be 55 pM. A K,,.~a, of 57/tM for gentisate at saturating [02] is derived from intercept replots with 02 as the fixed changing substrate. The estimated turnover number at saturating oxygen concentration is approximately 24,000 rain- t (per active site). Under turnover conditions, gentisate 1,2-dioxygenase is rapidly inactivated at oxygen concentrations above 800 aM. Substrate Specificity and Inhibition. Gentisate 1,2-dioxygenase from P. acidovorans catalyzes the turnover of a range of alkyl- and halo-substituted gentisates. Somewhat reduced rates are observed with gentisate containing substitutions (methyl, ethyl, 2-propyl, bromo, fluoro) at the ring C-3 position. Analogs of gentisate with similar substitutions in either the C-4 or in t8 j. C. Salerno and J. N. Siedow, Biochim. Biophys. Acta 579, 246 (1979).
[18]
SYNTHESIS OF DIHYDROXY AROMATIC COMPOUNDS
107
both the C-3 and C-4 positions are also turned over, but at dramatically reduced rates (< 10% that ofgentisate). All three main functional groups of gentisate appear to be required for efficient turnover, in that little or no turnover is observed with compounds in which any one of these groups is substituted with another functionality. Additionally, monosubstituted benzoates such as salicylate, 3-hydroxybenzoate, and thiosalicylate are not turned over but are relatively good inhibitors of the enzyme. Gentisate 1,2-dioxygenase is inactivated in a time- and concentration-dependent manner by oxidants such as H202 and K3Fe(CN)6. Partial reactivation is effected by ascorbate. Acknowledgment This work was supported by a grant from the National Institutes of Health (GM 24689).
[18] S y n t h e s i s o f 170- o r 1 s O - E n r i c h e d D i h y d r o x y Aromatic Compounds B y A L L E N M . ORVILLE, M A R K R . H A R P E L , a n d JOHN D . LIPSCOMB
Specific isotopic labeling of the oxygen atoms of phenolic and catecholic compounds is of use in spectroscopic and kinetic investigations of a variety of enzymes that bind these molecules as substrates or inhibitors.t.2 A convenient and versatile synthetic procedure is depicted in Scheme 1. The position of incorporation of the isotopically enriched oxygen atom is determined by the source of the enriched oxygen (H20 or 02) and the position of substituents in the starting aromatic amine. This methodology is illustrated here with the synthesis of isotopically enriched homoprotocatechuate (3,4-dihydroxyphenylacetate, HPCA a) as an example. This procedure has appeared in a previous report. 2 These techniques have been shown to be applicable to a wide variety of phenolic and catecholic aromatic compounds (Table I).
1D. M. Arciero and J. D. Lipscomb, J. Biol. Chem. 261, 2170 (1986). 2 A. M. Orville and J. D. Lipscomb, J. Biol. Chem. 264, 8791 (1989). 3 Abbreviations used are given in Table I.
METHODS IN ENZYMOLOGY,VOL. 188
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