©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Methionine Salvage Pathway in Klebsiella pneumoniae and Rat Liver
IDENTIFICATION AND CHARACTERIZATION OF TWO NOVEL DIOXYGENASES (*)

(Received for publication, October 12, 1994; and in revised form, November 28, 1994)

Jonathan W. Wray (§) Robert H. Abeles (¶)

From the Institute of Molecular Biology, University of Oregon, Eugene, Oregon, 97403

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The 5-methylthio-D-ribose moiety of 5`-(methylthio)-adenosine is converted to methionine in a wide variety of organisms. 1,2-Dihydroxy-3-keto-5-methylthiopentene anion (an aci-reductone) is an advanced intermediate in the methionine salvage pathway present in the Gram-negative bacterium Klebsiella pneumoniae and rat liver. This metabolite is oxidized spontaneously in air to formate and 2-keto-4-methylthiobutyric acid (the alpha-keto acid precursor of methionine). Previously, we had purified an enzyme (E2) from Klebsiella which catalyzes the oxidative degradation of the aci-reductone to formate, CO, and methylthiopropionic acid. To further characterize the reactions of the aci-reductone we used its desthio analog, 1-2-dihydroxy-3-ketohexene anion (III), which was described previously. This molecule undergoes the analogous enzymatic and non-enzymatic reactions of the natural substrate, namely the formation of formate, CO, and butyrate from III. Experiments with ^18O(2) show that E2 is a dioxygenase which incorporates one molecule of ^18O into formate and butyric acid. No cofactor has been identified. We were unable to find an enzyme which catalyzes the conversion of 1,2-dihydroxy-3-keto-5-methylthiopentane to a keto acid precursor of methionine. The keto acid is probably produced non-enzymically in Klebsiella. We have, however, identified and purified an enzyme (E3) from rat liver, which catalyzes the formation of formate and 2-oxopentanoic acid from III. This enzyme has a monomeric molecular mass of 28,000 daltons, and no chromophoric cofactor has been identified. Experiments with ^18O(2) show that E3 is a dioxygenase which incorporates an ^18O molecule into formate and the alpha-keto acid. In rat liver CO formation was not detected.


INTRODUCTION

5`-Methylthioadenosine (MTA), (^1)a metabolite derived from S-adenosylmethionine, is converted to methionine in a wide variety of organisms ((1) , and references therein) The pathway by which methionine is recycled in the Gram-negative bacterium Klebsiella pneumoniae, as well in rat liver, has been investigated in our laboratory and elsewhere. In the first step of this conversion, methythioadenosine 1 (Fig. S1) is hydrolyzed by a specific nucleosidase to methylthioribose 2 and adenine(2, 3) . Methythioribose is phosphorylated at the expense of ATP by a specific kinase to produce 1-phosphomethylthioribose 3 as the alpha-anomer(4, 5) (bacteria and mammals). This glycosyl phosphate is then isomerized by aldose-ketose isomerase to 1-phospho-methylthioribulose 4. Ketose phosphate 4 is dehydrated to give (following ketonization) 2,3-diketo-5-methylthio-1-phosphopentane 5(6) .


Scheme 1: Scheme 1The conversion of methylthioadenosine to methionine.



The metabolism of the diketone 5 is intriguing. In order for its transformation to methionine to be complete, this molecule must undergo an oxidative carbon-carbon bond cleavage to the alpha-keto acid 8, which is then transaminated to methionine. We have isolated and characterized a bifunctional enzyme, designated E1, from K. pneumoniae, which first enolizes the diketone to the phosphoene-diol 6, then dephosphorylates this compound to the aci-reductone 7(1) . In K. pneumoniae this metabolite represents a branch point in the methionine salvage pathway, in that it can be converted to 8 and formate, or to CO, formate and methylthiopropionic acid(7) .

The work described here was undertaken in order to characterize the enzyme(s) in K. pneumoniae involved in the metabolism of 7. For comparison the metabolism of 7 in rat liver was also investigated.


MATERIALS AND METHODS

Substrate Analogs

The characterizations of the reaction(s) of the aci-reductone 7 requires substantial quantities of this compound. We have been unable to develop a viable chemical synthesis of 7 or the diketone 5, probably due to the presence of the methylthio substituent at C-4. This substituent predisposes the diketone to beta-elimination and oxidation. Previously we have reported the synthesis of the desthio analogs of compounds 7 and 5 (compounds I and III), and shown them to be alternate substrates for the reactions undertaken by the bacterial enzymes of the methionine salvage pathway (1) These reactions are shown in Fig. S2.


Scheme 2: Scheme 2The reactions of compounds I and III.



Enzyme Assay

Enzyme activity, both E2 and E3, was identified by the disappearance of the substrate III as monitored by the decrease in its characteristic absorbance at 305 nm. III was generated in situ by the action of the enolase-phosphotase (E1) from K. pneumoniae on compound I. The reaction mixture (0.5 ml) included 1.5 mMI, 0.5 mM MgSO(4), 5 µg of catalase, 5 µg of E1 (0.1 unit, 1 unit = 1 µmol of formate formed from I/min/mg protein), 50 mM KP(i), pH 7.5, at 25 °C.

Enzyme Purification

The purification of E2 from K. pneumoniae is described elsewhere(7) . The purification of the rat liver enzyme E3 is as follows. Frozen rat livers (40 g) (Pel-Freez), in 100 ml 0.5 mM MgSO(4), 1.0 mM dithiothreitol, 0.5 M KP(i), pH 7.5, were homogenized in a Waring blender 2 times for 30 s at 4 °C. The homogenate was centrifuged at 37,000 times g for 20 min then at 22,000 times g for an additional 40 min. To the supernatant fluid (107 ml) was added dropwise with stirring 57 ml of acetone at -20 °C over 15 min while maintaining the temperature between -7 and -10 °C, by means of a dry ice glycerol bath. The suspension was centrifuged at 14,000 times g for 50 min in precooled tubes at -10 °C. To the supernatant fluid (140 ml) 110 ml of acetone (-20 °C) were added as described above. After centrifugation the precipitate was taken up in 0.5 mM MgSO(4), 1.0 mM dithiothreitol, 50 mM KP(i), pH 7.5 (buffer A), to a final volume of 30 ml and dialyzed twice against 2 liters of buffer A for 10 h. Any insoluble material was removed by centrifugation at 14,000 times g for 30 min at 4 °C. To the supernatant fluid (45 ml) was added ammonium sulfate (14.7 g) to 55% saturation. The suspension was stirred for 30 min at 4 °C and then centrifuged at 22,000 times g for 30 min at 4 °C. The precipitate was taken up in buffer A to a final volume of 10 ml and dialyzed twice against 1 liter of buffer A for 10 h. The protein solution (total final volume 18 ml) was applied in 6-ml batches onto a Bio-Gel P-60 column (100-200 mesh, 2.5 times 40 cm, Bio-Rad). The column was equilibrated and eluted with buffer A. Fractions (5 ml) were collected. The active fractions, 16 and 17, were pooled and chromatographed on a Pharmacia Biotech Inc. fast protein liquid chromatography apparatus. To 5 ml of protein solution 2.2 ml of a saturated solution of ammonium sulfate in buffer A were added, and the solution was applied onto a phenyl-Superose 5/5 column (Pharmacia) equilibrated with 1.2 M (NH(4))(2)SO(4) in buffer A (buffer B). At a flow rate of 0.4 ml the column was eluted with 4 ml of buffer B, then 4 ml of 100 to 60% buffer B in a linear gradient, then 30 ml of 60 to 0% buffer B in a linear gradient. Fractions (0.8 ml) nos. 25 and 26 containing enzyme activity were combined. SDS-PAGE of E3 indicated a purity of >90%. Table 1shows the results of a typical purification.



Synthesis of I

The synthesis of I from 2-oxopentanoic acid is described in Myers et al.(1) .

Formate Assay

The reaction, in a total volume of 1 ml, contained 50 mM KP(i), pH 7.5, 0.5 mM MgSO(4), 0.15 mM compound III, and enzyme to be assayed. The reaction proceeded for 15 min at 37 °C. The reaction was stopped by heating for 2 min in a boiling water bath and then cooled to 4 °C. To the cooled solution were added 20 µl of NAD (50 mM) and 20 µl (34 units) of formate dehydrogenase (20 mg/ml in 50 mM KP(i), pH 7.5). The reaction was monitored at 340 nm and was allowed to go to completion.

O(2) Uptake

Measurement was by means of an O(2) electrode (Hansatech Instruments) at 30 °C, in a total volume of 0.5 ml. The reaction mixture contained 50 mM KP(i) pH 7.5, 0.5 mM MgSO(4), 0.15 mM compound III, and enzyme to be assayed.

alpha-Keto Acid Assay

The reaction (total volume 1.0 ml) contained 250-500-µl aliquots of the reaction mix to be assayed, 500-750 µl of 50 mM KP(i), pH 7.5, 10 liters of 10 µM NADH. The reaction was started by the addition of 100 units (10 µl) of bovine heart lactate dehydrogenase (Sigma). Absorbance was monitored at 340 nm. The reaction was allowed to go to completion.

Determination of Organic Acids

Analysis was done by HPLC monitored by a refractive index detector. A Waters organic acid column (300 times 7.8 mm) was used and eluted with 5 mM H(2)SO(4) at a flow rate of 0.5 ml/min.

Conversion of III to Products in the Presence of ^18O(2)

E1 and catalase in 1.0 ml of buffer, 50 mM KP(i), 0.5 mM MgSO(4), pH 7.5) were degassed for 15 min using a water aspirator at 4 °C. The reaction vessel was then purged with argon and stirred for an additional 15 min under a stream of argon. The cycle of degassing and argon flushing was repeated. Compound I was then added to the reaction mix (final concentration 8 mM), and the reaction was allowed to proceed for 15 min to allow for the conversion of I to III. The reaction was started by the addition of enzyme (E2 or E3) in degassed buffer, followed by the addition of 98.3% ^18O(2) to a pressure of 1 atm. The reaction was allowed to proceed for 20 min followed by quenching the reaction by the addition of 50 µl of 2 M HClO(4) to precipitate the protein. The protein was removed by centrifugation in a bench top centrifuge, and the reaction mixture was brought to pH 9.0 by the addition of sodium hydroxide. The reaction mixture was then lyophilized and the salts removed by precipitation by the addition of ethanol. The organic acid products were derivatized and subjected to analysis by mass spectrometry.

Derivatization of Organic Acids

The following modification of the method of Hutchinson and Mabuini (8) was employed. The sodium salt of the organic acid reaction products, formate, oxopentanoate, and butyrate, was reacted under an atmosphere of nitrogen, with a 2-fold excess of p-phenylphenacyl bromide and 1 eq of 18-crown-6 in 1:1 (v/v) dry acetonitrile:benzene at reflux for 24 h. The suspension was evaporated to dryness using a stream of nitrogen. The resulting residue was taken up in methylene chloride, applied onto a PrepSep-Si normal phase silica extraction column (Fischer Scientific), and eluted with methylene chloride (2 ml) then diethyl ether (5 ml). The combined eluates were evaporated under a stream of nitrogen, and the residue was taken up in toluene (1 ml) for purification by HPLC.

HPLC of Derivatized Acids

HPLC purification of the derivatized acids was done using a column (10 times 250 mm) of LiChrosphere Si-100 10-µm spherical normal phase silica gel (Merck) eluted with toluene at 1.0 ml/min. Peak detection, A nm, was accomplished using an Isco V^4 absorbance detector. One-minute fractions were collected, and those containing pure p-phenylphenacyl-derivatized acids were pooled. The solvent was evaporated to dryness under a stream of nitrogen. Derivatized reaction products co-eluted with authentic p-phenylphenacylbutyrate, p-phenylphenacylformate, and p-phenylphenacyloxopentanoate. The purity of the derivatized reaction products was determined by ^1H NMR spectroscopy. ^1H NMR data (CdCl(3)), (ppm). For p-phenylphenacyl formate: 8.28 (s, 1H, -O(2)C H), 8.01 (td, 2H, aromatic), 7.73 (td, 2H, aromatic), 7.64 (td, td, 2H, aromatic), 7.50-7.40 (m, 3H, aromatic), and 5.48 (s, 2H, -COCH(2)O(2)CH). For p-phenylphenacyl butyrate: 8.05 (td, 2H, aromatic), 7.75 (td, 2H, aromatic), 7.68 (td, td, 2H, aromatic), 7.55-7.45 (m, 3H, aromatic), 5.38 (s, 2H, -COCH(2)OR), 2.52 (t, 2H, -COCH(2)R), 1.77 (m, 2H, COCH(2)CH(2)CH(3)), and 1.02 (t, 3H, COCH(2)CH(2)CH(3)). For p-phenylphenacyloxopentanoate: 7.80 (td, 2H, aromatic), 7.52 (td, 2H, aromatic), 7.45 (td, td, 2H, aromatic), 7.38-7.26 (m, 3H, aromatic), 5.31 (s, 2H, -COCH(2)OR), 2.70 (t, 2H, -COCOC H(2)R), 1.53 (m, 2H, COCOCH(2)C H(2)CH(3)), and 0.78 (t, 3H, COCOCH(2)CH(2)CH(3)).

Mass Spectrometry of Derivatized Reaction Products

Mass spectrometry was performed using electron or chemical ionization with a Finnegan-Mat 90 spectrometer.

Cofactor Analysis

The absence of a chromophoric cofactor of E2 led us to investigate the existence of a metal or dissociable cofactor by a variety of methods. Incubation of E2 with the iron-specific chelating agents alpha,alpha`-dipyridyl (10M), O-phenanthroline (10M), and 1,2-dihydroxy-benzene-3,5-disulfonate (10M) showed no more than 8% inhibition. Compounds such as ethylxanthate (10M), toluene-3,4-dithiol (10M), and diethyldithiocarbamate (10M) that are highly specific for copper were also ineffective as inhibitors(9) .

E2 was concentrated to 40 µM in 20 mM Tris-HCl, pH 7.0, and submitted for electrospray mass spectrometry. Protein under native conditions 1:1 buffer, 0.2% acetic acid, gave a mass ion of 20,196 ± 10. Protein run under denaturing conditions, 1:1 buffer, 50% acetonitrile, 5% acetic acid, gave identical mass ion.

Atomic absorption spectroscopy was performed to look for iron and zinc. Metal determinations were performed by electrothermal atomic absorption spectroscopy with a Perkin-Elmer model 4100 ZL instrument. Protein, prepared as describe for electrospray mass spectrometry, was diluted in 0.2% metal-free nitric acid (Baker). Aliquots (10 µl) of sample were loaded into pyroltically coated graphite tubes with an AS-70 automatic sampling device. The metal and zinc content is less than 0.01 g atom/mol of enzyme.


RESULTS

Purification and Characterization of E2 from K. pneumoniae

In partially purified extracts of K. pneumoniaeIII is converted to formate and 2-ketopentanoic acid, with concomitant O(2) uptake(1) . We have previously reported that the oxidation of III can proceed non-enzymically and is inhibited by catalase(1, 7) . Oxidation of III by crude bacterial extracts in the presence of catalase is not completely abolished. The oxidation of III must, therefore be partially enzymatic. We wanted to isolate and characterize the enzyme(s) responsible for the oxidation of III. In order to accomplish this we inhibited the non-enzymic oxidation of III in bacterial extracts with catalase and then searched for a protein fraction which can overcome this inhibition. We made the assumption that the enzymic oxidation of III is not inhibited by catalase. This strategy proved successful. We isolated, and purified to homogeneity (as judged by SDS-PAGE), an enzyme which catalyzes the oxidation of III. This enzyme will be referred to as E2. The enzyme is monomeric and has a molecular weight of 20,000. No evidence for the presence of a cofactor was obtained.

Reaction Stoichiometry

The stoichiometry of the reaction was examined. The reaction mixture contained 95 nmol of compound III, 5 µg of E2, 0.5 mM MgSO(4), 50 mM KP(i), pH 7.5, in a total volume of 0.5 ml (standard assay conditions). The reaction was allowed to proceed until O(2) uptake ceased. At this point 90 nmol of O(2) were consumed. A simultaneous formate assay (formate dehydrogenase) determined 73 nmol of formate. HPLC analysis (organic acid column) identified butyrate (92 nmol), and confirmed formate (84 nmol) as reaction products. To further identify the reaction products, a large scale enzymic reaction (5.0 mM compound III) was performed and the products generated were examined by ^1H NMR. Analysis of the product mixture confirmed the enzymatic production of formate and butyrate. The ^1H NMR spectrum of the enzymatic reaction products contained a singlet at = 8.47 ppm which was assigned to HCOOK (compare standard HCOONa = 8.46 ppm). The spectrum also contained the following signals: 1) = 2.17 ppm (triplet); 2) = 1.56 ppm (multiplet); 3) = 0.90 ppm (triplet). These signals are in excellent agreement with authentic sodium butyrate. No evidence for the generation of any other compound was provided by ^1H NMR.

To further characterize the reaction, [C-1-^3H]III (specific activity, 75,000 cpm/µmol) was prepared(7) . E2 was added to isotopic III under standard assay conditions. Examination of the reaction mixture by HPLC showed 50% of added radioactivity as formate, 40% in H(2)O, and 5% as an unidentified compound. Formate isolated by HPLC had a specific activity of 78,000 cpm/mol. (^2)No radioactivity was found in butyric acid. There results establishes that formate is formed primarily, possibly exclusively, from C-1 of III.

The stoichiometry of the reaction requires formation of another ``1-C'' compound. It is likely that this compound is derived from C-2 of III. To identify this compound we synthesized [2-^14C]III (specific activity, 30,000 cpm/µmol). Upon the action of enzyme on [2-^14C]III no radioactivity was found in the quenched reaction mixture. We concluded that the compound derived from C-2 of III is a volatile, water insoluble compound. We have identified this molecule by its reaction with deoxyhemoglobin as carbon monoxide (7) . The reaction was carried out under standard assay conditions in a gas-tight cuvette. Upon completion of the reaction deoxyhemoglobin was added with a syringe, and the mixture was scanned between 500-650 nm and 370-450 nm. The absorption maximum of deoxyhemoglobin at 555 nm disappeared, and new absorption maxima at 569 and 539 nm appeared. The Soret band shifted from 430 to 419 nm (Fig. 1, A and B). These data indicate the binding of carbon monoxide to deoxyhemoglobin(10) . The absorption spectrum for carbomonoxyhemoglobin remains until the ratio of deoxyhemoglobin to compound III exceeds one, at which point the deoxyhemoglobin spectrum begins to emerge as dominant. Concentration of the reaction mixture, followed by gel filtration chromatography determined that the ^14C-moiety is associated with hemoglobin. This result establishes C-2 of III as the source of CO, and that C-2 is not converted to any other reaction products. It is concluded that E2 catalyzes as shown in Fig. R1.


Figure 1: Identification of CO. The reaction mixture, in an air tight cuvette, in a total volume of 0.5 ml: 0.15 mM III, 0.5 mM MgSO(4), 50 mM KP(i), pH 7.4. E2 (4.8 times 10 units) was added with a syringe through a septum. The reaction was monitored at 305 nm, the absorbance maximum of III. After the spectrum returned to base line, 11 µM deoxyhemoglobin was added. The spectrum of the reaction mixture was scanned before and after the addition of deoxyhemoglobin. A, spectrum between 500 and 650 nm; B, spectrum between 370 and 450 nm. (- -) deoxyhemoglobin;(- - -) deoxyhemoglobin added to the reaction mixture.




Figure R1: Reaction 1.



^18O(2) Incorporation

To identify the site of dioxygen attack in the enzyme catalyzed oxidation of III, the reaction was carried out in ^18O(2). The mass spectra of the derivatized reaction products show that one atom of ^18O(2) was incorporated into formate and butyrate (Table 2). A second ^18O(2) experiment was done and the infra red spectrum of carbomonoxyhemoglobin was determined. The spectrum was consistent with that of O(2) carbomonoxyhemoglobin, i.e. no ^18O incorporation into CO occurs(11) .



Metabolism of I and III in Rat Liver

The action of rat liver homogenates on [^14C-2] compound I, Fig. S2(the preparation of which is described in (1) and (7) ) was examined. To 150 nmol of ^14C-2 compound I, 50 mM KP(i), 0.5 mM MgSO(4), pH 7.5, was added 0.5 mg of protein in a total volume of 0.5 ml. The reaction was allowed to proceed for 30 min and quenched with 2 M HClO(4). The diketone was converted to alpha-ketopentanoic acid (28%), 65% remained as the unconverted diketone, 7% of the radioactivity was found as unidentified products. [^14C]ketopentanoate and I were identified by HPLC chromatography using the organic acids column as described in the methods section. By analogy the natural substrate 5 (Fig. S1) would be converted to the alpha-keto acid precursor of methionine. This experiment established that the diketone 5 is a likely intermediate in the methionine salvage pathway in rat liver, and that 7 is an intermediate in the conversion of 5 to 8. We therefore investigated the metabolism of III in rat liver. As above, we fractionated rat liver homogenate in order to find a protein fraction which would catalyze the oxidation of III in the presence of catalase, which inhibits non-enzymic oxidation of III. Such a fraction was found and this activity was purified to near homogeneity, >90% as indicated by SDS-PAGE. The purified protein has a molecular mass of 29,000 daltons. No spectral evidence for the presence of a cofactor was obtained. The stoichiometry of this enzymatic reaction, however, was different than that of E2. E2 catalyzes the formation of CO, formate, and butyrate from I, whereas this enzyme (E3) catalyzes the formation of formate and oxopentanoic acid from I. The reaction mixture contained 100 nmol of compound III generated under standard assay conditions. The reaction was allowed to proceed until O(2) uptake ceased. At this time, 93 nmol of O(2) were consumed. A simultaneous formate assay determined 78 nmol of formate. HPLC analysis identified, 2-keto pentanoate (89 nmol), and confirmed formate (80 nmol) as reaction products. E3 catalyzes as shown in Fig. R2.


Figure R2: Reaction 2.



^18O(2) Incorporation

The action of E3 on compound III was examined using ^18O(2) as the oxygen source in order to determine the site of dioxygen attack. The data from the mass spectrometry of the derivatized reaction products show that a single ^18O(2) atom appears in formate and the carboxyl group of 2-oxopentanoate (Table 2).

The studies described above were done with compound III, an analog of 1,2-dihydroxy-5-methylthiopentene anion (7, Fig. S1). We carried out experiments to determine whether the natural metabolite is a substrate for E2, and E3. Compound 7 (80 nmol) was enzymatically prepared as described previously (1) and was added to 40 µg of E2 in an oxygen electrode under our standard assay conditions. Total O(2) consumption was 75 nmol; 68 nmol of methylthiopropionic acid and and 67 nmol of formate were produced. A parallel experiment examined the action of E3 on compound 7. O(2) consumption was 73 nmol, 60 nmol of alpha-keto acid, and 68 nmol of formate were generated. These data sugest that the natural metabolite, 1,2-dihydroxy-5-methylthiopentene anion, is a substrate for both E2 and E3.


DISCUSSION

In the methionine salvage pathway S-methylthioadenosine (MTA) is converted to methionine. We had previously reported that MTA is converted to 2,3-diketo-5-methylthio-1-phosphopentane 5 which, in turn, is converted to the aci-reductone 7 (Fig. S1). The final conversion of 7 to the alpha-keto acid precursor of methionine probably occurs non-enzymically in K. pneumoniae. We have also purified an enzyme from K. pneumoniae which converts 7 to CO, formate, and beta-S-methylthiopropionic acid. All enzymes and intermediates of the methionine salvage pathway in K. pneumoniae have now been identified.

In rat liver MTA is also converted to methionine, it has been established that 5 is a likely metabolite in the conversion of MTA to methionine(12) . We have isolated and purified an enzyme from rat liver which converts 5 to the alpha-keto acid precursor of methionine. No corresponding enzyme is found in K. pneumoniae. The results reported here strongly suggest that the methionine salvage pathway in rat liver involves similar reactions as in K. pneumoniae. In the course of this work we have isolated two dioxygenases E2 and E3. Some information concerning their mechanism of action has been obtained.

Carbon monoxide forming enzymes are rare. To date the only mammalian carbon monoxide-forming enzyme is heme oxygenase, which is operative in the pathway of heme degradation(13) . This enzyme acts on protohemin IX to form biliverdin and carbon monoxide. Three other carbon monoxide-forming enzymes have been identified in the fungus Aspergillus flavus and in the bacteria Pseudomonas putida and Arthrobacter sp. Ru61a. These enzymes are dioxygenases which are involved in heterocyclic ring cleavage reactions (14, 15, 16) .

Quercertinase from A. flavus is a copper-containing dioxygenase which oxidatively cleaves the ring of quercertin to yield carbon monoxide and depside (2-protocatechuoylphloroglucinolcarboxylic acid). When ^18O(2) is used as the dioxygen source, the enzyme incorporates one ^18O molecule into the 2 and 4 positions of the depside, but not into carbon monoxide. When [3-^14C]quercertin was used as a substrate, all of the radioactivity was present in the carbon of carbon monoxide(17) . The product distribution of the carbon monoxide-forming enzyme from K. pneumoniae (E2) is analogous to that of quecertinase. Recently studies of the CO-forming dioxygenase from the bacterium Arthrobacter sp. Ru61a have indicated an analogous reaction to that of E2, in which no metal or chromophoric cofactor has been identified(18) .

The reaction of E2 represents a 1,3-oxygenolytic attack which results in the oxidative cleavage of two carbon-carbon bonds. A proposed mechanism for the formation of carbon monoxide from compound III is described in Fig. 2. This mechanism necessitates the formation of a five membered cyclic peroxide intermediate, which then decomposes to form products. Fig. 2represents the addition of oxygen to the carbanion tautomer of the aci-reductone to form the hydroperoxide anion which then attacks the C-3 carbonyl. The reactions of carbanions with molecular oxygen have been well studied(19) . An investigation of the autoxidation of ketone and ester anions by Gersmann and Bickel (20) have shown that the primary products of these reactions are alpha-hydroperoxides, which can be isolated in high yields. The formation of the peroxide intermediate has also been proposed by Abell and Schloss (21) as an intermediate in oxygenase side reactions of carbanion forming enzymes. They determined that the expression of oxygenase activity was dependent on the accessibility of the carbanionic intermediate to molecular oxygen and on the ability of the enzyme to stabilize the initially formed peroxide anion through protonation with an enzymic group or through metal coordination.


Figure 2: Proposed mechanism of E2.



There is some reluctance, however, to accept the simple one-step mechanism for the formation of the hydroperoxy anion from the reaction of a carbanion with molecular oxygen, as it involves a violation of the spin conservation rule(22) . Instead, a stepwise single electron transfer from the carbanion to molecular oxygen could be proposed for the formation of the hydroperoxy anion intermediate. Carbon monoxide is a readily extruded functional group in many reactions and its extrusion from the cyclic peroxide intermediate may proceed by a radical, or a concerted mechanism(23, 24) .

E3 is a dioxygenase which also utilizes the aci-reductone as a substrate. E3, however, directs a 1,2-oxygenolytic attack on the substrate. A speculative reaction mechanism is depicted in Fig. 3. This mechanism suggests the formation of a dioxetane as an intermediate which subsequently decomposes to form products. The reaction to form the alkyperoxide intermediate from the attack of the carbanion on O(2) is the same as that described for E2. The reaction differs from that of E2 in that the alkyperoxide anion intermediate adds to C-2 to form the dioxetane, rather than C-3 to form the five-membered cyclic peroxide. The fact that this enzyme, as well as E2, is not inhibited by catalase suggests that an alkylperoxide intermediate is not released into solution.


Figure 3: Proposed mechanism of E3.




CONCLUSION

The metabolism of the aci-reductone 7 has been investigated in both the K. pneumoniae and rat liver. This compound is metabolized enzymatically in rat liver by a dioxygenase to the alpha-keto acid precursor of methionine. This enzyme represents the final enzymatic transformation of the methionine salvage pathway which needed to be identified. In K. pneumoniae this metabolite represents a branch point in the pathway, whose metabolism can follow two possible routes. This molecule can either undergo a non-enzymatic oxidation to the alpha-keto acid precursor of methionine, or can be acted upon by the CO-forming enzyme to yield CO, formate, and methylthiopropionic acid. Recent experiments in mammals, which establish CO as a neurotransmitter(25) , invite interesting speculation of its possible role as diffusible messenger in bacteria.


FOOTNOTES

*
This work was supported by National Science Foundation Grant DMB-8920779 (to R. H. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by Brandeis Macro Molecular Structure and Mechanism Training Grant GM07596.

To whom correspondence should be addressed: Dept. of Biochemistry, Brandeis University, 415 South St., Waltham, MA 02154. Tel.: 617-736-2310; Fax: 617-736-2349.

(^1)
The abbreviations used are: MTA, S-methylthioadenosine; PAGE, polyacrylamide gel electrophoresis; HPLC, high peformance liquid chromatography.

(^2)
[C-1-^3H] III is enzymatically generated from [C1-^3H] I (specific activity, 150,000 cpm/µmol). This generation is shown in Fig. R3. In the conversion of C-1-labeled I to III, 50% of the tritum originally in I should be found in solvent water, and the specific activity of [C-1-^3H]III should be 75,000 cpm/µmol.


Figure R3: Reaction 3.




ACKNOWLEDGEMENTS

We thank Drs. Vallee and Shapiro of Harvard University for the atomic absorption spectroscopy, Dr. Creech of Boston University for the mass spectrometry, and Dr. Karen Wong of The Sandoz Research Institute for the electrospray mass spectrometry. We also thank Jana Johnson for help in preparation of this manuscript. This is publication no. 1779 of the Graduate Department of Biochemistry, Brandeis University.


REFERENCES

  1. Myers, R. W., Wray, J. W., Fish, S., and Abeles, R. H. (1993) J. Biol. Chem. 268, 24785-24791 [Abstract/Free Full Text]
  2. Duerre, J. A. (1962) J. Biol. Chem. 237, 3737-3741 [Free Full Text]
  3. Ferro, A. J., Barrett, A., and Shapiro, S. K. (1976) Biochim. Biophys. Acta 438, 487-494 [Medline] [Order article via Infotrieve]
  4. Gianotti, A. J., Tower, P. A., Sheley, J. H., Conte, P. A., Spiro, C., Ferro, A. J., Fitchen, J. H., and Riscoe, M. K. (1990) J. Biol. Chem. 265, 831-837 [Abstract/Free Full Text]
  5. Ferro, A. J., Barrett, A., and Shapiro, S. K. (1978) J. Biol. Chem. 253, 6021-6025 [Abstract]
  6. Furfine, E. S., and Abeles, R. H. (1988) J. Biol. Chem. 263, 9598-9606 [Abstract/Free Full Text]
  7. Wray, J. W., and Abeles, R. H. (1993) J. Biol. Chem. 268, 21466-21469 [Abstract/Free Full Text]
  8. Hutchinson, C. R., and Mabuni, C. T. (1977) J. Labelled Compd. Radiopharm. 13, 571-574
  9. Oka, T., and Simpson, F. J. (1971) Biochem. Biophys. Res. Commun. 43, 1-5 [Medline] [Order article via Infotrieve]
  10. Waterman, M. R. (1978) Methods Enzymol. 52, 456-463 [Medline] [Order article via Infotrieve]
  11. Maxwell, J. C., and Caughey, W. S., (1978) Methods Enzymol. 54, 302- 323 [Medline] [Order article via Infotrieve]
  12. Trackman, P. C., and Abeles, R. H. (1981) Biochem. Biophys. Res. Commun. 103, 1238-1244 [Medline] [Order article via Infotrieve]
  13. Tenhunen, R., Marver, H. S., and Schmid, R. (1969) J. Biol. Chem. 244, 6388-6394 [Abstract/Free Full Text]
  14. Westlake, D. W. S., Talbot, G., Blakley, E. R., and Simpson, F. J. (1959) Can. J. Microbiol. 5, 621-628
  15. De Beyer, A., and Lingens, F. (1993) Biol. Chem. Hoppe-Seyler 374, 101-110 [Medline] [Order article via Infotrieve]
  16. Bott, G., Schmidt, M., Rommel, T. O., and Lingens, F. (1990) Biol. Chem. Hoppe-Seyler 371, 999-1003 [Medline] [Order article via Infotrieve]
  17. Simpson, F. J., Talbot, G., and Westlake, D. W. S. (1960) Biochem. Biophys. Res. Commun. 2, 15-22 [Medline] [Order article via Infotrieve]
  18. Bauer, I., De Beyer, A., Tshisuaka, B., Fetzner, S., and Lingens, F. (1994) FEMS Microbiol. Lett. 117, 299-304
  19. Barton, and Jones (1965) J. Chem. Soc. Pt. III 3563-3570
  20. Gersmann, H. R., and Bickel, A. F. (1971) J. Chem. Soc. 12c, 2230-2237
  21. Abell, L. M., and Schloss, J. V. (1991) Biochemistry 30, 7883-7887 [Medline] [Order article via Infotrieve]
  22. Russel, G. A. (1968) Adv. Chem. Sci. 75, 174-180
  23. Tsuji, and Ohno (1969) Synthesis 157-169
  24. Stark, B. P., and Duke, A. J. (1967) Extrusion Reactions , pp. 16-46, Pergamon, Oxford
  25. Verma, A., Hirsch, D. J., Glatt, C. E., Ronnett, G. V., and Snyder, S. H. (1993) Science 259, 381-384 [Medline] [Order article via Infotrieve]

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