- Email: [email protected]
Vanillate hydroxylase from Sporotrichum pulverulentum
274
[28]
LIGNIN
compounds.7,8Thus, NAD(P)H: quinone oxidoreductases may play a role in reversing any enzymatic or nonenzymatic conversion of phenols within the fungal cytoplasm, thereby avoiding inhibitory effects and ensuring further conversion of the benzenoid compounds to intermediates of central metabolism. Pyridine nucleotide: quinone reductase systems may also participate in electron transfer between respiratory substrates and polyphenol oxidases. 9 R. F. Bilton and R. B. Cain, Biochem. J. 108, 829 (1968). J. M. Varga and H. Y. Neujahr, Acta Chem. Scand. 26, 509 (1972). 9 W. D. Wosilait, N. Nason, and A. J. Terrell, J. Biol. Chem. 206, 271 (1954).
[28] V a n i l l a t e H y d r o x y l a s e f r o m S p o r o t r i c h u m pulverulentum B y J O H N A. BUSWELL a n d K A R L - E R I K ERIKSSON
Vanillate hydroxylase1-3 catalyzes the oxidative decarboxylation of vanillic acid to 2-methoxyhydroquinone (Scheme 1). Activity of the enzyme may be determined by measuring (1) 14CO2evolution from p4C-carboxyl]vanillic acid, (2) 02 consumption using the oxygen electrode, (3) the decrease in absorbance at 340 nm resulting from the oxidation of the NADH or NADPH cofactor, or (4) methoxyhydroquinone production by gas- liquid chromatography. Assay Methods
1. Enzyme Assay Based on ~4C02 Evolution from [14C]Carboxyl.labeled Vanillic Acid Reagents Potassium phosphate buffer, 100 mM, pH 7.4 NAD(P)H, 10 mM, prepared fresh daily, 0.3 ml [~4C-carboxyl]Vanillic acid (6.2 X 106 dpm/mg dissolved in absolute ethanol, 29 nmol in 5 #1 (26,000 dpm) Enzyme protein, 0.1-0.3 ml J. A. Buswell, P. Ander, B. Pettersson, and K.-E. Eriksson, FEBSLett. 103, 98 (1979). 2 j. A. Buswell, K.-E. Eriksson, and B. Pettersson, J. Chromatogr. 215, 99 (1981). 3 y. Yajima, A. Enoki, M. B. Mayfield, and M. H. Gold, Arch. Microbiol. 123, 319 (1979).
METHODSIN ENZYMOLOGY,VOL. 161
Col~'ight© 1988by AcademicPrem,Inc. Allrightsof re0roductionin any formreserved.
[28]
VANILLATE HYDROXYLASE FROM S.
+ " ~ H ~OCH3
NAD(P)H + 0 2
"
6
pulverulentum
275
+ CO z + N A D ( P I + H20 CH 3
SCHEME 1. Reaction scheme for vanillate hydroxylase.
Procedure. Potassium phosphate buffer, 200 pmol, enzyme protein (crude extract or purified enzyme), and NAD(P)H, 3 pmol, in a total volume of 3.0 ml in a 125-ml conical flask are equilibriated at 30 ° in a shaker water bath. In cases where crude extract is used, reduced pyridine nucleotide is added to the reaction vessel just prior to initiation of the reaction by addition of radiolabeled vanillic acid substrate. This is to avoid extensive oxidation of cofactor which may occur due to NAD(P)H oxidase activity in the extracts. After addition of substrate, flasks are tightly sealed with rubber stoppers from which small glass tubes, containing 1 ml 1 N NaOH to absorb 1aco2, are suspended. 4 At appropriate intervals, the NaOH is transferred into scintillation vials. The glass tubes are rinsed twice with 0.5 ml H20 and the washings also added to the vial. To this 2 ml is added 10 ml Picofluor 30, containing 1% Carbosorb (Packard), and the vials allowed to stand for at least 1 hr at 4 ° before measuring radioactivity in a scintillation counter. 4 To improve the efficiency of this technique in terms of quantitative rate determinations of enzyme activity, rubber stoppers can be fitted with a flushing device and ~4CO2 air-flushed directly into vials containing the scintillation cocktail. 2. Enzyme Assay Based on Oxygen Uptake Measurements Reagents Potassium phosphate buffer, 100 mM, pH 7.4 NAD(P)H, 10 mM, 0.1 ml Vanillic acid in distilled water adjusted to pH 6.8 with 1 N NaOH, 0.1 ml Enzyme protein, 0.1-0.3 ml Procedure. Potassium phosphate buffer, 250 gmol, enzyme protein, and NAD(P)H, 1.0 gmol, are added in a total volume of 2.9 ml to the reaction vessel of a Clark oxygen electrode (Rank, Bottisham, England). After equilibration at 30 °, oxygen consumption is measured following addition of 1/tmol (in 0.1 ml) vanillate and corrected for oxygen uptake in the absence of substrate.
4 p. Ander, A. Hatakka, and K.-E. Eriksson, Arch. Microbiol. 125, 189 (1980).
276
LlOrqlN
[28]
3. Enzyme Assay Based on Spectrophotometric Measurement Reagents. As for assay (2) above. Procedure. Potassium phosphate buffer, 250/lmol, enzyme protein, and NAD(P)H, 1.0 /tmol, are added in a total volume of 2.9 ml to a cuvette of 1.0-cm light path. After equilibration at 30 °, 1.0/tmol (in 0.1 ml) vanillate is added and the rate of NAD(P)H oxidation measured from the decrease in absorbance at 340 nm. Values are corrected for NAD(P)H oxidation in the absence of substrate. 4. Enzyme Assay Based on Methoxyhydroquinone Production Reagents Potassium phosphate buffer, 100 mM, pH 7.4 Vanillic acid, 10 m M NAD(P)H, 10 m M Enzyme protein Procedure. Potassium phosphate buffer (7 ml), enzyme protein (0.11.0 ml), and vaniUic acid (1.0 ml), in a total volume of 10 ml in a 125-ml conical flask, are equilibrated at 30 ° in a shaker water bath. The reaction is initiated by addition of 20/tmol NAD(P)H (in 2.0 ml) and allowed to proceed for I hr before acidifying with 0.5 ml concentrated HCI. Precipitated protein is removed by centrifugation; syringol is added as an internal standard and the supernatant is extracted three times with 10 ml diethyl ether. Combined ethereal extracts are dried over anhydrous Na2SO4 and, after evaporating off the ether in a stream of nitrogen, the residue taken up in 0.4 ml dry pyridine. An aliquot (100/tl) is silylated with bistrimethylsilyltrifluoroacetamide (BSTFA) for 1 hr at room temperature and methoxyhydroquinone determined by gas-liquid chromatography using a Packard model 427 with flame-ionization detector. Identification is based on comparison with the retention time of authentic methoxyhydroquinone. Separation is achieved using a glass capillary column SE-30 (25 m × 0.36 mm) and the following operating conditions: injection 220", detection 250 °, program 8 min, 150 °, rise 6°/min to 170 °, final time 7 min. Definition of E n z y m e Unit and Specific Activity One unit of vanillate hydroxylase is the amount which converts 1 pmol of vanillic acid into methoxyhydroquinone and carbon dioxide per minute at 30 °. Specific activity is expressed as U/mg protein, as determined by the method of Bradford, 5 with bovine serum albumin as the protein standard. 5 M. M. Bradford, Anal. Biochem. 72, 248 0976).
[28]
VANILLATE HYDROXYLASE FROM S.
pulverulentum
277
Purification P r o c e d u r e
Growth of the Fungus Sporotrichum pulverulentum (ATCC 32629 anamorph of Phanerochaete chrysosporium) is grown in shake culture at 28 ° in l-liter conical flasks containing 300 ml of a modified Norkrans' medium ~to which 3 m M vanillate is added. A spore suspension6 (2.5 × 106 spores) serves as inoculum and mycelial pellets are harvested after 50 hr and may be used either immediately or stored frozen at - 2 0 ° until required. The purification procedure, carried out at 0 - 5 ° unless otherwise stated, involves four steps starting with the fungal mycelium: (1) preparation of crude extract and precipitation with potassium phosphate, (2) fractionation on a phenyl-Sepharose bed, (3) chromatofocusing, and (4) affinity chromatography on phenyl-Sepharose. Step I: Preparation of Crude Extract. A total of approximately 120 g wet weight of fresh or frozen mycelium is suspended in 4 vol 0.1 M KH2PO4-NaOH buffer (pH 7.4) and broken in separate batches in a 50-ml homogenizer (Thomas Co., Philadelphia, PA) for 3 min. Combined homogenates are clarified by centrifugation at 30,000 g for 30 rain. Following dialysis against 0.001 M potassium phosphate buffer (pH 7.4), the enzyme solution is concentrated by freeze drying and, if necessary, may be stored at - 20 ° in this form. The freeze-dried material is dissolved in 0.1 M potassium phosphate buffer (pH 7.0) so that a total absorbance at 280 nm o f - 9 5 0 0 is obtained. An equal volume of 2.0 M potassium phosphate buffer (pH 7.0) is then added and the solution slowly stirred at room temperature for 30 min. The precipitate is removed by centrifugation and the supernatant retained. Step 2: Phenyl-SepharoseChromatography. Supernatant material from step 1 is passed through a phenyl-Sepharose bed, 40 × 50 m m (Pharmacia, Uppsala, Sweden), equilibriated with 1.0 M potassium phosphate buffer (pH 7.0) at a rate of 8 ml/min. Under these conditions, vanillate hydroxylase is quantitatively retained on the phenyl-Sepharose. The bed is washed with 1.0 M potassium phosphate buffer (pH 7.0) and then successively with 500 ml each of 0.5 and 0.25 M potassium phosphate buffer until the absorbance reading of the washing solution at 280 nm is zero in both cases. The enzyme is finally eluted from the phenyl-Sepharose bed with a mixture of 0.2 M potassium phosphate buffer (pH 7.0) and an equal amount of ethylene glycol. The pooled active fractions are dialyzed against distilled water for 2 hr to remove the ethylene glycol and then concentrated by 6 K.-E. Eriksson and S. C. Johnsrud, Enzyme Microbiol. Technol. 5, 425 (1983).
278
LI6NIN
[28]
ultrafiltration using an Immersible Molecular Separator (Millipore, Bedford, MA) to a total volume of 15 ml. Step 3: Chromatofocusing. After desalting on a PD-10 column (Pharmacia), the concentrated enzyme solution is applied to a PBE 94 (Pharmacia) column (200 × 9 mm) previously equilibriated with 200 ml of 20 m M Tris buffer (pH 8.0). The column is then eluated with Polybuffer 96 until adjusted to pH 6.0 with glacial acetic acid. Step 4: Affinity Chromatography on Phenyl-Sepharose-Vanillic Acid Gel. Phenyl-Sepharose-vanillic acid gel is prepared by adding a solution of vanillic acid (200 mg) in acetone (20 ml) to 13 g phenyl-Sepharose gel suspended in 50 ml of acetone. The gel is kept in suspension by end-overend rotation and diluted stepwise with water, allowing 1 hr between each dilution step for equilibration. The acetone concentration is thereby reduced sequentially from 100% to 66, 33, 17, and 8%. After the last step, the acetone is removed by washing with water and the gel finally equilibriated with 1 M potassium phosphate buffer (pH 7.0). Active fractions from step 3 are pooled and concentrated to 1.5 ml by ultrafiltration using Immersible Molecular Separators. The concentrated enzyme is applied to a phenyl-Sepharose-vaniUic acid column (120 × 6 mm) and eluted with a linear gradient (total volume 200 ml), simultaneously decreasing from 1.0 to 0.05 M potassium phosphate (pH 7.0) and increasing from 0 to 50% ethylene glycol. Purified enzyme protein gives a single band when examined using analytical isoelectric focusing and sodium dodecyl sulfate gel electrophoresis. The chemicals, apparatus, preparation of gels, and technique for isoelectric focusing are as described by Vesterberg7 and Ayers et aL8 Data from a typical preparation of vanillate hydroxylase, resulting in an approximately 240-fold purification and an overall yield of 13.3%, are summarized in Table I.
Properties General Based on protein molecular weight standards, the molecular weight of vanillate hydroxylase is estimated to be 65,000. In crude mycelial extracts the enzyme is active in potassium phosphate buffer over a wide pH range (5.8-8.0) and activity peaks are observed at pH 6.6 and 7.8. In common with other aromatic hydroxylases, vanillate hydroxylase activity is markedly reduced in Tris-HCl buffer (40% inhibition compared with activity in potassium phosphate at pH 7.2). Addition of 0.1 M KC1 or NaCI to assay mixtures using potassium phosphate buffer reduces enzyme 70. Vesterberg, Biochim. Biophys. Acta 257, 11 (1972). s A. R. Ayers, S. B. Ayers, and K.-E. Edksson, Eur. J. Biochem. 90, 171 (1978).
I=I 0 c,,I ~1
xx
x
~'~
~,
< ,...I
;,... x 0
I~ ,.-., ~a
M ~
0 Z
£
~e~. ~1
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g
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'-4
8
~ ' S , .~ t~ ~
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=1
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LIGNIN
[28]
activity by approximately 70%, indicating that inhibition is probably due to chloride ions. Cofactor Supplementation. Both NADH and NADPH serve as electron donors for vanillate hydroxylase although enzyme activity with NADH is only about 85% of that observed with NADPH using the 14CO2 evolution assay. Activity is stimulated still further when FAD is used in combination with NADPH but not NADH, although there is no evidence of a flavin component being associated with the enzyme. Substrate Specificity. Several substrate analogs promote NADH oxidation by partially purified vaniUate hydroxylase, indicating the enzyme to be specific for phenolic compounds with a hydroxyl group located para to a carboxyl substituent attached directly to the aromatic ring. NADH oxidation proceeds at about the same rate when vanillate is replaced by either protocatechuate, p-hydroxybenzoate, or 2,4-dihydroxybenzoate. Gallate, 3-O-methylgallate, 2,4,6-trihydroxybenzoate, and 2,3,4-trihydroxybenzoate also promote high rates of NADH oxidation although activity with syringate is ~ 10% compared to vanillate. Inhibitors. Tiron and heavy metals (Cu 2+, Ag 2+, Hg2+) at 1 m M concentrations and 0. l mMp-chloromercuribenzoate completely inhibit vanillate hydroxylase. Inactivation by the latter is partially reversible by addition of stoichiometric amounts of reduced glutathione or dithiothreitol. Cyanide (1 mM) and a,ot'-dipyridyl (1 mM) also depress enzyme activity by about 15 and 39%, respectively, but arsenite, azide, EDTA, and diethyl dithiocarbamate at 1 m M concentrations have no significant inhibitory effect. Stoichiometry. Vanillate hydroxylase is presumed to catalyze a typical monooxygenase reaction although the exact stoichiometry is difficult to establish since the reaction product, methoxyhydroquinone, undergoes nonenzymatic oxidation to the corresponding quinone, which in turn is reduced by any excess of NADPH present in the reaction mixture. Thus, observed oxygen uptake is usually slightly more than ascribed to a monooxygenase reaction although the rate of quinone formation is relatively slow at slightly acidic pH values and the presence of crude or partially purified fungal extract retards the rate of nonenzymatic oxidation even further. However, vanillate hydroxylase oxidatively decarboxylates protocatechuate and 2,4-dihydroxybenzoate to hydroxyquinol which, in turn, undergoes intradiol ring cleavage to maleylacetate. Ring fission is catalyzed by a dioxygenase present in crude mycelial extracts. In reaction mixtures containing 100-200 nmol of protocatechuate or 2,4-dihydroxybenzoate and crude extract, consumption of 2 nmol oxygen/nmol of substrate is observed. By analogy, oxidative decarboxylation of vanillate consumes 1 nmol of oxygen/nmol vanillate converted to methoxyhydroquinone. The
[29]
4-METHOXYBENZOATE MONOOXYGENASE
281
stoichiometry of NADPH consumption is not available due to the asymptotic nature of the decrease in absorbance at 340 nm. Role and Distribution of Enzyme. Vanillic acid is found in relatively high yield in extracts of wood following fungal decay) It is a breakdown product of the lignin component and a catabolic intermediate in the degradation of lignin-related compounds by white rot fungi and other microorganisms) TM Vanillate hydroxylase is found in many brown rot and white rot fungi and oxidative decarboxylation via methoxyhydroquinone may serve as the major route for vanillic acid catabolism in these two groups of wood-decaying fungi)2,13 9 E. Adler, WoodSci. Technol. 11, 169 (1977). 10j. K. Gupta, S. G. Hamp, J. A. Buswell, and K.-E. Eriksson, Arch. Microbiol. 128, 349 (1981). " M. Ohta, T. Higuchi, and S. Iwahara, Arch. Microbiol. 121, 23 (1979). 12T. K. Kirk and L. F. Lorenz, Appl. Microbiol. 26, 173 (1973). i; j. A. Buswell, K.-E. Eriksson, J. K. Gupta, S. G. Hamp, and I. Nordh, Arch. Microbiol. 131, 366 (1982).
[29] 4 - M e t h o x y b e n z o a t e
Monooxygenase
from
P s e u d o m o n a s putida: I s o l a t i o n , B i o c h e m i c a l P r o p e r t i e s , Substrate Specificity, and Reaction Mechanisms Enzyme Components
of the
B y F R I T H J O F - H A N S BERNHARDT, E C K H A R D BILL, A L F R E D X A V E R TRAUTWEIN, and H A N S TWILFER
Importance of 4-Methoxybenzoate Monooxygenase in Bacterial Metabolism Various authors who have investigated the biological degradation of lignanes as model substances for lignin have pointed out that certain soil fungi belonging to the Basidiomycetes and Ascomycetes are able to use the plant structural substance lignin as a carbon source. 1-4 Lignin, which is highly polymerized and water insoluble, is a major component of plant residues which are degraded in the soil by microorganisms. In the degradation of lignin by soil microorganisms, the cleavage of intramolecular aryl-alkyl ethers plays a central role. Studies on the mechai W. F. Van Vliet, Biochim. Biophys. Acta 15, 211 (1954). 2 H. lshikawa, W. J. Schubert, and F. F. Nord, Arch. Biochem. Biophys. 100, 131 (1963). 3 T. K. Kirk, W. J. Connors, R. D. Bleam, W. F. Hackett, and J. G. Zeikus, Proc. Natl. Acad. Sci. U.S.A. 72, 2515 (1975). 4 T. K. Kirk, W. J. Connors, and J. G. Zeikus, Appl. Environ. Microbiol. 32, 192 (1976).
METHODSIN ENZYMOLOGY,VOL. 161
Copyright© 1988by AcademicPress,Inc. Allfightsof reproductionin any formreserved.
[28]
LIGNIN
compounds.7,8Thus, NAD(P)H: quinone oxidoreductases may play a role in reversing any enzymatic or nonenzymatic conversion of phenols within the fungal cytoplasm, thereby avoiding inhibitory effects and ensuring further conversion of the benzenoid compounds to intermediates of central metabolism. Pyridine nucleotide: quinone reductase systems may also participate in electron transfer between respiratory substrates and polyphenol oxidases. 9 R. F. Bilton and R. B. Cain, Biochem. J. 108, 829 (1968). J. M. Varga and H. Y. Neujahr, Acta Chem. Scand. 26, 509 (1972). 9 W. D. Wosilait, N. Nason, and A. J. Terrell, J. Biol. Chem. 206, 271 (1954).
[28] V a n i l l a t e H y d r o x y l a s e f r o m S p o r o t r i c h u m pulverulentum B y J O H N A. BUSWELL a n d K A R L - E R I K ERIKSSON
Vanillate hydroxylase1-3 catalyzes the oxidative decarboxylation of vanillic acid to 2-methoxyhydroquinone (Scheme 1). Activity of the enzyme may be determined by measuring (1) 14CO2evolution from p4C-carboxyl]vanillic acid, (2) 02 consumption using the oxygen electrode, (3) the decrease in absorbance at 340 nm resulting from the oxidation of the NADH or NADPH cofactor, or (4) methoxyhydroquinone production by gas- liquid chromatography. Assay Methods
1. Enzyme Assay Based on ~4C02 Evolution from [14C]Carboxyl.labeled Vanillic Acid Reagents Potassium phosphate buffer, 100 mM, pH 7.4 NAD(P)H, 10 mM, prepared fresh daily, 0.3 ml [~4C-carboxyl]Vanillic acid (6.2 X 106 dpm/mg dissolved in absolute ethanol, 29 nmol in 5 #1 (26,000 dpm) Enzyme protein, 0.1-0.3 ml J. A. Buswell, P. Ander, B. Pettersson, and K.-E. Eriksson, FEBSLett. 103, 98 (1979). 2 j. A. Buswell, K.-E. Eriksson, and B. Pettersson, J. Chromatogr. 215, 99 (1981). 3 y. Yajima, A. Enoki, M. B. Mayfield, and M. H. Gold, Arch. Microbiol. 123, 319 (1979).
METHODSIN ENZYMOLOGY,VOL. 161
Col~'ight© 1988by AcademicPrem,Inc. Allrightsof re0roductionin any formreserved.
[28]
VANILLATE HYDROXYLASE FROM S.
+ " ~ H ~OCH3
NAD(P)H + 0 2
"
6
pulverulentum
275
+ CO z + N A D ( P I + H20 CH 3
SCHEME 1. Reaction scheme for vanillate hydroxylase.
Procedure. Potassium phosphate buffer, 200 pmol, enzyme protein (crude extract or purified enzyme), and NAD(P)H, 3 pmol, in a total volume of 3.0 ml in a 125-ml conical flask are equilibriated at 30 ° in a shaker water bath. In cases where crude extract is used, reduced pyridine nucleotide is added to the reaction vessel just prior to initiation of the reaction by addition of radiolabeled vanillic acid substrate. This is to avoid extensive oxidation of cofactor which may occur due to NAD(P)H oxidase activity in the extracts. After addition of substrate, flasks are tightly sealed with rubber stoppers from which small glass tubes, containing 1 ml 1 N NaOH to absorb 1aco2, are suspended. 4 At appropriate intervals, the NaOH is transferred into scintillation vials. The glass tubes are rinsed twice with 0.5 ml H20 and the washings also added to the vial. To this 2 ml is added 10 ml Picofluor 30, containing 1% Carbosorb (Packard), and the vials allowed to stand for at least 1 hr at 4 ° before measuring radioactivity in a scintillation counter. 4 To improve the efficiency of this technique in terms of quantitative rate determinations of enzyme activity, rubber stoppers can be fitted with a flushing device and ~4CO2 air-flushed directly into vials containing the scintillation cocktail. 2. Enzyme Assay Based on Oxygen Uptake Measurements Reagents Potassium phosphate buffer, 100 mM, pH 7.4 NAD(P)H, 10 mM, 0.1 ml Vanillic acid in distilled water adjusted to pH 6.8 with 1 N NaOH, 0.1 ml Enzyme protein, 0.1-0.3 ml Procedure. Potassium phosphate buffer, 250 gmol, enzyme protein, and NAD(P)H, 1.0 gmol, are added in a total volume of 2.9 ml to the reaction vessel of a Clark oxygen electrode (Rank, Bottisham, England). After equilibration at 30 °, oxygen consumption is measured following addition of 1/tmol (in 0.1 ml) vanillate and corrected for oxygen uptake in the absence of substrate.
4 p. Ander, A. Hatakka, and K.-E. Eriksson, Arch. Microbiol. 125, 189 (1980).
276
LlOrqlN
[28]
3. Enzyme Assay Based on Spectrophotometric Measurement Reagents. As for assay (2) above. Procedure. Potassium phosphate buffer, 250/lmol, enzyme protein, and NAD(P)H, 1.0 /tmol, are added in a total volume of 2.9 ml to a cuvette of 1.0-cm light path. After equilibration at 30 °, 1.0/tmol (in 0.1 ml) vanillate is added and the rate of NAD(P)H oxidation measured from the decrease in absorbance at 340 nm. Values are corrected for NAD(P)H oxidation in the absence of substrate. 4. Enzyme Assay Based on Methoxyhydroquinone Production Reagents Potassium phosphate buffer, 100 mM, pH 7.4 Vanillic acid, 10 m M NAD(P)H, 10 m M Enzyme protein Procedure. Potassium phosphate buffer (7 ml), enzyme protein (0.11.0 ml), and vaniUic acid (1.0 ml), in a total volume of 10 ml in a 125-ml conical flask, are equilibrated at 30 ° in a shaker water bath. The reaction is initiated by addition of 20/tmol NAD(P)H (in 2.0 ml) and allowed to proceed for I hr before acidifying with 0.5 ml concentrated HCI. Precipitated protein is removed by centrifugation; syringol is added as an internal standard and the supernatant is extracted three times with 10 ml diethyl ether. Combined ethereal extracts are dried over anhydrous Na2SO4 and, after evaporating off the ether in a stream of nitrogen, the residue taken up in 0.4 ml dry pyridine. An aliquot (100/tl) is silylated with bistrimethylsilyltrifluoroacetamide (BSTFA) for 1 hr at room temperature and methoxyhydroquinone determined by gas-liquid chromatography using a Packard model 427 with flame-ionization detector. Identification is based on comparison with the retention time of authentic methoxyhydroquinone. Separation is achieved using a glass capillary column SE-30 (25 m × 0.36 mm) and the following operating conditions: injection 220", detection 250 °, program 8 min, 150 °, rise 6°/min to 170 °, final time 7 min. Definition of E n z y m e Unit and Specific Activity One unit of vanillate hydroxylase is the amount which converts 1 pmol of vanillic acid into methoxyhydroquinone and carbon dioxide per minute at 30 °. Specific activity is expressed as U/mg protein, as determined by the method of Bradford, 5 with bovine serum albumin as the protein standard. 5 M. M. Bradford, Anal. Biochem. 72, 248 0976).
[28]
VANILLATE HYDROXYLASE FROM S.
pulverulentum
277
Purification P r o c e d u r e
Growth of the Fungus Sporotrichum pulverulentum (ATCC 32629 anamorph of Phanerochaete chrysosporium) is grown in shake culture at 28 ° in l-liter conical flasks containing 300 ml of a modified Norkrans' medium ~to which 3 m M vanillate is added. A spore suspension6 (2.5 × 106 spores) serves as inoculum and mycelial pellets are harvested after 50 hr and may be used either immediately or stored frozen at - 2 0 ° until required. The purification procedure, carried out at 0 - 5 ° unless otherwise stated, involves four steps starting with the fungal mycelium: (1) preparation of crude extract and precipitation with potassium phosphate, (2) fractionation on a phenyl-Sepharose bed, (3) chromatofocusing, and (4) affinity chromatography on phenyl-Sepharose. Step I: Preparation of Crude Extract. A total of approximately 120 g wet weight of fresh or frozen mycelium is suspended in 4 vol 0.1 M KH2PO4-NaOH buffer (pH 7.4) and broken in separate batches in a 50-ml homogenizer (Thomas Co., Philadelphia, PA) for 3 min. Combined homogenates are clarified by centrifugation at 30,000 g for 30 rain. Following dialysis against 0.001 M potassium phosphate buffer (pH 7.4), the enzyme solution is concentrated by freeze drying and, if necessary, may be stored at - 20 ° in this form. The freeze-dried material is dissolved in 0.1 M potassium phosphate buffer (pH 7.0) so that a total absorbance at 280 nm o f - 9 5 0 0 is obtained. An equal volume of 2.0 M potassium phosphate buffer (pH 7.0) is then added and the solution slowly stirred at room temperature for 30 min. The precipitate is removed by centrifugation and the supernatant retained. Step 2: Phenyl-SepharoseChromatography. Supernatant material from step 1 is passed through a phenyl-Sepharose bed, 40 × 50 m m (Pharmacia, Uppsala, Sweden), equilibriated with 1.0 M potassium phosphate buffer (pH 7.0) at a rate of 8 ml/min. Under these conditions, vanillate hydroxylase is quantitatively retained on the phenyl-Sepharose. The bed is washed with 1.0 M potassium phosphate buffer (pH 7.0) and then successively with 500 ml each of 0.5 and 0.25 M potassium phosphate buffer until the absorbance reading of the washing solution at 280 nm is zero in both cases. The enzyme is finally eluted from the phenyl-Sepharose bed with a mixture of 0.2 M potassium phosphate buffer (pH 7.0) and an equal amount of ethylene glycol. The pooled active fractions are dialyzed against distilled water for 2 hr to remove the ethylene glycol and then concentrated by 6 K.-E. Eriksson and S. C. Johnsrud, Enzyme Microbiol. Technol. 5, 425 (1983).
278
LI6NIN
[28]
ultrafiltration using an Immersible Molecular Separator (Millipore, Bedford, MA) to a total volume of 15 ml. Step 3: Chromatofocusing. After desalting on a PD-10 column (Pharmacia), the concentrated enzyme solution is applied to a PBE 94 (Pharmacia) column (200 × 9 mm) previously equilibriated with 200 ml of 20 m M Tris buffer (pH 8.0). The column is then eluated with Polybuffer 96 until adjusted to pH 6.0 with glacial acetic acid. Step 4: Affinity Chromatography on Phenyl-Sepharose-Vanillic Acid Gel. Phenyl-Sepharose-vanillic acid gel is prepared by adding a solution of vanillic acid (200 mg) in acetone (20 ml) to 13 g phenyl-Sepharose gel suspended in 50 ml of acetone. The gel is kept in suspension by end-overend rotation and diluted stepwise with water, allowing 1 hr between each dilution step for equilibration. The acetone concentration is thereby reduced sequentially from 100% to 66, 33, 17, and 8%. After the last step, the acetone is removed by washing with water and the gel finally equilibriated with 1 M potassium phosphate buffer (pH 7.0). Active fractions from step 3 are pooled and concentrated to 1.5 ml by ultrafiltration using Immersible Molecular Separators. The concentrated enzyme is applied to a phenyl-Sepharose-vaniUic acid column (120 × 6 mm) and eluted with a linear gradient (total volume 200 ml), simultaneously decreasing from 1.0 to 0.05 M potassium phosphate (pH 7.0) and increasing from 0 to 50% ethylene glycol. Purified enzyme protein gives a single band when examined using analytical isoelectric focusing and sodium dodecyl sulfate gel electrophoresis. The chemicals, apparatus, preparation of gels, and technique for isoelectric focusing are as described by Vesterberg7 and Ayers et aL8 Data from a typical preparation of vanillate hydroxylase, resulting in an approximately 240-fold purification and an overall yield of 13.3%, are summarized in Table I.
Properties General Based on protein molecular weight standards, the molecular weight of vanillate hydroxylase is estimated to be 65,000. In crude mycelial extracts the enzyme is active in potassium phosphate buffer over a wide pH range (5.8-8.0) and activity peaks are observed at pH 6.6 and 7.8. In common with other aromatic hydroxylases, vanillate hydroxylase activity is markedly reduced in Tris-HCl buffer (40% inhibition compared with activity in potassium phosphate at pH 7.2). Addition of 0.1 M KC1 or NaCI to assay mixtures using potassium phosphate buffer reduces enzyme 70. Vesterberg, Biochim. Biophys. Acta 257, 11 (1972). s A. R. Ayers, S. B. Ayers, and K.-E. Edksson, Eur. J. Biochem. 90, 171 (1978).
I=I 0 c,,I ~1
xx
x
~'~
~,
< ,...I
;,... x 0
I~ ,.-., ~a
M ~
0 Z
£
~e~. ~1
.<
g
>
'-4
8
~ ' S , .~ t~ ~
~' ~= .~ o ' ~
=1
280
LIGNIN
[28]
activity by approximately 70%, indicating that inhibition is probably due to chloride ions. Cofactor Supplementation. Both NADH and NADPH serve as electron donors for vanillate hydroxylase although enzyme activity with NADH is only about 85% of that observed with NADPH using the 14CO2 evolution assay. Activity is stimulated still further when FAD is used in combination with NADPH but not NADH, although there is no evidence of a flavin component being associated with the enzyme. Substrate Specificity. Several substrate analogs promote NADH oxidation by partially purified vaniUate hydroxylase, indicating the enzyme to be specific for phenolic compounds with a hydroxyl group located para to a carboxyl substituent attached directly to the aromatic ring. NADH oxidation proceeds at about the same rate when vanillate is replaced by either protocatechuate, p-hydroxybenzoate, or 2,4-dihydroxybenzoate. Gallate, 3-O-methylgallate, 2,4,6-trihydroxybenzoate, and 2,3,4-trihydroxybenzoate also promote high rates of NADH oxidation although activity with syringate is ~ 10% compared to vanillate. Inhibitors. Tiron and heavy metals (Cu 2+, Ag 2+, Hg2+) at 1 m M concentrations and 0. l mMp-chloromercuribenzoate completely inhibit vanillate hydroxylase. Inactivation by the latter is partially reversible by addition of stoichiometric amounts of reduced glutathione or dithiothreitol. Cyanide (1 mM) and a,ot'-dipyridyl (1 mM) also depress enzyme activity by about 15 and 39%, respectively, but arsenite, azide, EDTA, and diethyl dithiocarbamate at 1 m M concentrations have no significant inhibitory effect. Stoichiometry. Vanillate hydroxylase is presumed to catalyze a typical monooxygenase reaction although the exact stoichiometry is difficult to establish since the reaction product, methoxyhydroquinone, undergoes nonenzymatic oxidation to the corresponding quinone, which in turn is reduced by any excess of NADPH present in the reaction mixture. Thus, observed oxygen uptake is usually slightly more than ascribed to a monooxygenase reaction although the rate of quinone formation is relatively slow at slightly acidic pH values and the presence of crude or partially purified fungal extract retards the rate of nonenzymatic oxidation even further. However, vanillate hydroxylase oxidatively decarboxylates protocatechuate and 2,4-dihydroxybenzoate to hydroxyquinol which, in turn, undergoes intradiol ring cleavage to maleylacetate. Ring fission is catalyzed by a dioxygenase present in crude mycelial extracts. In reaction mixtures containing 100-200 nmol of protocatechuate or 2,4-dihydroxybenzoate and crude extract, consumption of 2 nmol oxygen/nmol of substrate is observed. By analogy, oxidative decarboxylation of vanillate consumes 1 nmol of oxygen/nmol vanillate converted to methoxyhydroquinone. The
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4-METHOXYBENZOATE MONOOXYGENASE
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stoichiometry of NADPH consumption is not available due to the asymptotic nature of the decrease in absorbance at 340 nm. Role and Distribution of Enzyme. Vanillic acid is found in relatively high yield in extracts of wood following fungal decay) It is a breakdown product of the lignin component and a catabolic intermediate in the degradation of lignin-related compounds by white rot fungi and other microorganisms) TM Vanillate hydroxylase is found in many brown rot and white rot fungi and oxidative decarboxylation via methoxyhydroquinone may serve as the major route for vanillic acid catabolism in these two groups of wood-decaying fungi)2,13 9 E. Adler, WoodSci. Technol. 11, 169 (1977). 10j. K. Gupta, S. G. Hamp, J. A. Buswell, and K.-E. Eriksson, Arch. Microbiol. 128, 349 (1981). " M. Ohta, T. Higuchi, and S. Iwahara, Arch. Microbiol. 121, 23 (1979). 12T. K. Kirk and L. F. Lorenz, Appl. Microbiol. 26, 173 (1973). i; j. A. Buswell, K.-E. Eriksson, J. K. Gupta, S. G. Hamp, and I. Nordh, Arch. Microbiol. 131, 366 (1982).
[29] 4 - M e t h o x y b e n z o a t e
Monooxygenase
from
P s e u d o m o n a s putida: I s o l a t i o n , B i o c h e m i c a l P r o p e r t i e s , Substrate Specificity, and Reaction Mechanisms Enzyme Components
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B y F R I T H J O F - H A N S BERNHARDT, E C K H A R D BILL, A L F R E D X A V E R TRAUTWEIN, and H A N S TWILFER
Importance of 4-Methoxybenzoate Monooxygenase in Bacterial Metabolism Various authors who have investigated the biological degradation of lignanes as model substances for lignin have pointed out that certain soil fungi belonging to the Basidiomycetes and Ascomycetes are able to use the plant structural substance lignin as a carbon source. 1-4 Lignin, which is highly polymerized and water insoluble, is a major component of plant residues which are degraded in the soil by microorganisms. In the degradation of lignin by soil microorganisms, the cleavage of intramolecular aryl-alkyl ethers plays a central role. Studies on the mechai W. F. Van Vliet, Biochim. Biophys. Acta 15, 211 (1954). 2 H. lshikawa, W. J. Schubert, and F. F. Nord, Arch. Biochem. Biophys. 100, 131 (1963). 3 T. K. Kirk, W. J. Connors, R. D. Bleam, W. F. Hackett, and J. G. Zeikus, Proc. Natl. Acad. Sci. U.S.A. 72, 2515 (1975). 4 T. K. Kirk, W. J. Connors, and J. G. Zeikus, Appl. Environ. Microbiol. 32, 192 (1976).
METHODSIN ENZYMOLOGY,VOL. 161
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