A New Ring Cleavage Enzyme : 2,3-Dihydroxybenzoate Oxygenase

80 A New Ring Cleavage Enzyme : 2,3-Dihydroxybenzoate Oxygenase DOUGLAS W. RIBBONS and ROBERT J. WATKINSON 1

Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: January 1, 1968 | doi: 10.1021/ba-1968-0077.ch080

Milstead Laboratory of Chemical Enzymology, "Shell" Research, Ltd., Broad Oak Road, Sittingbourne, Kent, England 2,3-Dihydroxybenzoate is oxidized by extracts of Pseudomonas fluorescens with the consumption of one mole of oxygen per mole of substrate. An equivalent amount of carbon dioxide is evolved, and α-hydroxymuconic semialdehyde is formed. Intermediates between 2,3-dihydroxybenzoate and α-hydroxymuconic semialdehyde have not been detected, nor has the site of ring cleavage been established. The enzyme is inactivated rapidly by air and other oxidants, but the activity may be restored by anaerobic incubation of the enzyme with reducing agents or Fe ions. The enzyme is inhibited by iron chelating agents such as α,α'-dipyridyl. 2+

2,3-Dihydroxybenzoate occurs as a metabolite of plants and micro organisms (8, 16). Certain fungi decarboxylate it to catechol after which it is oxygenated and converted by known pathways to 3-oxoadipate (11,14). Pseudomonas fluorescens, however, utilizes 2,3-dihydroxybenzoate as its sole carbon source and cleaves the benzenoid ring of this substrate ( 9 ) . The enzyme(s) catalyzing the oxygenation of 2,3-dihydroxybenzoate to α-hydroxymuconic semialdehyde is named 2,3-dihydroxybenzoate oxygenase; a preliminary report has described the stoichiometry of the reaction ( s ) ( 9 ). Extracts of Pseudomonas fluorescens 23D-1, grown in the presence of 2,3-dihydroxybenzoate, catalyze the rapid oxidation of 2,3-dihydroxybenzoate to a yellow acidic intermediate with the spectral characteristics of α-hydroxymuconic semialdehyde (2, 5), which is also the product of the oxidation of catechol by catechol 2,3-oxygenase. Carbon dioxide is also evolved during dihydroxybenzoate oxidation in amounts equivalent to those of the substrate added to the enzyme ( Table I ). Permanent address: Department of Biochemistry, School of Medicine, P. O. Box 875, Biscayne Annex, University of Miami, Miami, Fla. 33152. 1

252 Mayo; Oxidation of Organic Compounds Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

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Table I.

253

A New Enzyme

Stoichiometry of Gaseous Exchange during 2,3-Dihydroxybenzoate Oxidation

2,3-Dihydroxybenzoate Supplied, pmoles

Oxygen Consumed, μτηοΐββ

Carbon Dioxide Evolved, μτηοΐβδ

5 10 10

4.9 9.9 10.2

4.7 9.8 9.7

The product, α-hydroxymuconic semialdehyde, was isolated and characterized from a large scale incubation. Its elemental analysis, infra red, ultraviolet and visible absorption spectra, and its rapid decomposition to pyruvate by extracts of Pseudomonas aeruginosa T l (10) are all con sistent with this structure. Intermediates between 2,3-dihydroxybenzoate and a-hydroxymuconic semialdehyde have not been detected. It is evident, however, that cate chol is not an intermediate i n the formation of 2,3-dihydroxybenzoate since extracts catalyze neither its formation anaerobically from 2,3-dihy droxybenzoate nor its oxidation. In fact, catechol is an inhibitor of 2,3-dihydroxybenzoate oxygenase. The exclusion of catechol as inter mediate in this metabolic transformation leaves two possibilities: (a) decarboxylation and oxygenation of the benzene nucleus are simultaneous, yielding α-hydroxymuconic semialdehyde directly, or (b) oxygenation precedes decarboxylation, and a second oxo-acid is intermediate. Our results so far do not differentiate between these possibilities. When purified extracts are oxidizing 2,3-dihydroxybenzoate, the rates of oxygen consumption, carbon dioxide evolution, and a-hydroxymuconic semialdehyde formation cannot be distinguished ( Figure 1 ). W i t h either possibility, the site of ring cleavage remains to be determined. As seen in Figure 2, cleavage of the nucleus between carbon atoms 3 and 4 would yield an oxo-acid, which could decarboxylate at carbon atom 7 to yield α-hydroxymuconic semialdehyde. Cleavage of 2,3-dihydroxybenzoate between carbon atoms 1 and 2 would yield a dioxodicarboxylic acid that could decarboxylate at either end of the carbon chain to give a-hydroxy muconic semialdehyde with its carbon atoms differently derived. Stability of 2,3 -Dihydroxybenzoate Oxygenase The enzyme is usually unstable in air in both crude extracts and purified fractions. Loss of activity is attributed to oxidation and is fairly common to oxygenases of this type (13). The oxidation may be induced by oxygen itself since preparations are more stable if stored under nitro gen or in vacuo or by the oxidized form of some of the usual sulfhydryl-

Mayo; Oxidation of Organic Compounds Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

254

OXIDATION

O F ORGANIC

COMPOUNDS

III

12


Oxygen

Figure 1. 2,3-Dihydroxybenzoate oxidation by partially purified extracts of Pseudomonas fluorescens Each Warburg jiask contained: 20% KOH in the center well (0.2 ml.) during measurements of oxy gen consumption, or 2N H>SO>, in the second sidearm (0.2 ml.) for determining carbon dioxide evolved; 25mM 2,3-dihydroxybenzoate in sidearm (0.2 ml); 0.067 M-KHPO,, pH 7.1 (0.6 ml); and enzyme solution (1 ml. of a Ρ-300 eluate fraction). Oxygen consumption was followed in duplicate flasks, and acid was tipped into the others at the times indicated. Portions of the arrested reaction mixtures were taken to determine a-hydroxymu conic semialdehyde formation spectrophotometrically

protecting reagents such as mercaptoethanol. This is based on evidence that the rate of inactivation by mercaptoethanol is greatly accelerated by adding factor Β [which catalyzes the autoxidation of mercaptoethanol (7) (Figure 3 ) ] . Hydrogen peroxide has been suggested as the inactivator under similar circumstances ( 6 ) , but the product of factor Β catalyzed oxidation of mercaptoethanol is not hydrogen peroxide but water ( 7 ) . Activity of the enzyme may be restored by various reducing reagents, usually by preincubation under anaerobic conditions. The more successful of these has been anaerobic incubation with sodium borohydride, cysteine, F e , and ascorbate. Ascorbate w i l l also give quick reactivation by aerobic incubation which led to the inclusion of ascorbate in the assay system. The response of different batches of enzyme to reactivation by various reducing agents is quite variable. 2+


80.

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A New Enzyme

255


Purification of 2,3-Dihydroxybenzoate Oxygenase Table II shows the steps used i n purifying 2,3-dihydroxybenzoate oxygenase. The cells were harvested by centrifugation, washed with 0.01M tris buffer p H 7.0 and stored as cell pastes at —14°C. until required. Crude extracts were prepared by suspending cells i n 0.01M tris buffer p H 7.0 (0.5 gram wt. wt./ml.) and disrupting with an M S E 100 watt ultrasonic disintegrator for 2 minutes and centrifuging at 5000 g for 15 minutes. H i g h speed supernatants of extracts were prepared by centrifuging crude extracts at 100,000 g for 2 hours. 2,3-Dihydroxybenzoate was assayed at 30 °C. i n a fully automated Unicam SP800 spectrophotometer at 430 m/x. The reaction cuvettes contained: 0.067M phosphate buffer, p H 7.1 (2.5 m l . ) ; 25 m M 2,3-dihy droxybenzoate (20 /^liters); enzyme solution (as required but usually between 5 and 50 ^liters). Under these conditions the molar extinction coefficient of α-hydroxymuconic semialdehyde at 430 π\μ is 3.2 X 10 . The assays were conducted at 430 τημ because this is a more convenient wavelength when simultaneous measurements of oxygen consumption and product formation are made, although it is much less sensitive an assay than that used by Kojima et al. (5). ;{

Properties of 2,3-Dihydroxybenzoate Oxygenase The purest preparations obtained catalyze the formation of 7.3 /xmoles of α-hydroxymuconic semialdehyde/min./mg. protein after reac tivation with N a B H , anaerobically. 4

2,3-D i hydroxy benζoa te

Figure 2.

Possible routes of a-hydroxymuconic semialdehyde formation from 2,3dihydroxybenzoic acid


256

OXIDATION


1

O F ORGANIC

2 Hours

COMPOUNDS

III

3

Figure 3. Effect of oxidized and reduced forms of mercaptoethanol on 2,3-dihydroxybenzoate oxygenase activity Δ » enzyme alone A , enzyme + factor Β (10~ M), mercaptoethanol (3.9 X 10~ M) added as indicated O, enzyme + mercaptoethanol (3.9 X 10~ M) • , enzyme + factor Β (10~ M) + mercaptoethanol (3.9 X 10- M) The enzyme (P-300) eluate was incubated in 0.01M tris/HCl buffer pH 7.0, in air or in vacuo with I-raM solutions of either cysteine, HCl, or FeSO>, and assayed spectrophotometrically at the times indicated 6

S

S

6

S

Table II.

High Speed Supernatant (NH ) S0 ppt. (33-45%) Biogel P300 Hydroxylapatite D E A E Cellulose 4

1

2

Purification of 2,3-Dihydroxybenzoate Oxygenase Total Protein, mg.

Total Activity, pinoles/min.

Specific Activity, pmoles/min. /mg. of Protein

% Yield

1000

470

100

0.5

30 21 19

0.62 2.40 7.35

4

220 41 12

137 100 88° e

After reactivation with NaBH4.

The Km value for 2,3-dihydroxybenzoate is 7.5 X 10"°M. The enzyme was non-competitively inhibited by «,α'-dipyridyl ( K , = 6 X 10" A/) and was competitively inhibited by catechol ( K = 10 M ) . 4-Methylcatechol and 4-ethylcatechol both inhibit the reaction at 2 X 10" M. Neither phenol nor salicylate inhibit at 2 X 10" M. o-Phenanthroline ( 10 Μ ) and K C N ( 10" M ) inhibit the reaction. Inhibition by o-phenanfi

r ,

f

4

4

Γΐ

3


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257

A New Enzyme

throline, α,α'-dipyridyl and K C N is more complete when the inhibitors are preincubated with the enzyme rather than added to the assay system. Reactivation of 2,3 -Dihydroxybenzoate Oxygenase Some preparations of 2,3-dihydroxybenzoate oxygenase may be re activated by anaerobic incubation with N a B H , F e , ascorbate, cysteine, 2+


4

Figure 4. Time course of reactivation of 2,3dihydroxybenzoate oxygenase by cysteine and Fe 2+

The enzyme (P-300 eluate) was incubated in air or in vacuo with J-mM solutions of either cysteine, HCl, or FeSO and assayed spectrophotometrically at the times indicated. All enzyme incubations were per formed in 0.01 M tris IHCl buffer pH 7.0, and por tions were taken for the standard assay h


258

OXIDATION

O F

ORGANIC

COMPOUNDS

III

or glutathione. Figure 4, shows the reactivation with F e - and cysteine as a function of time. In some cases the effect of N a B H and F e - was additive. Of these reagents, only ascorbate induced reactivation when added to the assay system ( Figure 5 ). +


4

+

10' M 3

Ascorbate concentration

Figure 5. Effect of ascorbate concentration on 2,3-dihydroxybenzoate oxygenase activity. Ascorbate was added to the standard assay mixture

Our results indicate that 2,3-dihydroxybenzoate oxygenase is similar to other dioxygenases that yield oxo-acids—e.g., catechol 2,3-oxygenase (3) and 3,4-dihydroxyphenylacetic acid 2,3-oxygenase (1). A l l are unstable in air and inactivated by oxidizing agents; they are reactivated by reducing agents under anaerobic conditions and are inhibited by Fe chelating agents. Decarboxylating oxygenases have been described previously. Salicylate and anthranilate hydroxylases (12, 17), lysine oxygenase (3), lactate oxygenase (4), and arginine oxygenase (15) all oxygenate the carbon atom from which the carboxyl is lost. External electron donors are required for the two hydroxylases, but the substrates of the last three enzymes presumably reduce the second oxygen atom to water. In addition, 2,3-dihydroxybenzoate oxygenase catalyzes a second carbon-carbon scission of the substrate to yield an aliphatic product. A common mechanism for these decarboxylating oxygenases might exist; in the latter case both atoms of oxygen are incorporated into the product with consequent ring opening. L>+

Acknowledgments W e thank Richard Ruffell who contributed to earlier phases of this work and Thomas Gajdatsy for technical assistance.


80. RIBBONS AND wATKINSON A New Enzyme

259


Literature Cited (1) Adachi, K., Takeda, Y., Senoh, S., Kita, H., Biochim. Biophys. Acta 93, 483 (1964). (2) Dagley, S., Evans, W. C., Ribbons, D. W., Nature 188, 560 (1960). (3) Hayaishi, O., Bact. Rev. 30, 720 (1966). (4) Hayaishi, O., Sutton, W. B., J. Am. Chem. Soc. 70, 4809 (1957). (5) Kojima, Y., Itada, N., Hayaishi, O., J. Biol. Chem. 236, 2223 (1961). (6) Nozaki, M., Kojima, Y., Nakazawa, Y., Fujisawa, H., Ono, K., Kotani, S., Hayaishi, O., Yamamo, T., "Biological and Chemical Aspects of Oxy genases," p. 347, K. Bloch, O. Hayaishi, eds., Maruzen Co. Ltd., Tokyo, 1966. (7) Peel, J. L., Biochem.J.88, 296 (1963). (8) Pittard, A. J., Gibson, F., Doy, C. H., Biochim. Biophys. Acta 57, 290 (1962). (9) Ribbons, D. W., Biochem.J.99, 30P (1966). (10) Ribbons, D. W., J. Gen. Microbiol. 44, 221 (1966). (11) Shepherd, C. J., Villanueva, J. R., J. Gen. Microbiol. 20, vii (1964). (12) Taniuchi, H., Hatanaka, M., Kuno, S., Hayaishi, O., Nakajima, M., Kurihara, N., J. Biol. Chem. 239, 2204 (1964). (13) Taniuchi, H., Kojima, Y., Kanetsuna, F., Ochiai, H., Hayaishi, O., Bio chem. Biophys. Res. Comm. 8, 97 (1962). (14) Terui, G., Enatsu, T., Tokaku, H., J. Ferment. Technol. 31, 65 (1963). (15) Thoai, Ν. V., Olomucki, Α., Biochim. Biophys. Acta 59, 533 (1962). (16) Towers, G. Η. N., "Biochemistry of Phenolic Compounds," p. 249, J. B. Harborne, ed., Academic Press, London, 1964. (17) Yamamoto, S., Katagiri, M., Maeno, H., Hayaishi, O., J. Biol. Chem. 240, 3408 (1965). RECEIVED October 9, 1967.


A New Ring Cleavage Enzyme : 2,3-Dihydroxybenzoate Oxygenase

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