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Eugenio Notomista,
Paola Capasso, and
Alberto Di Donato*
Dipartimento di Biologia Strutturale e Funzionale, Università di Napoli Federico II, Complesso Universitario di Monte S. Angelo, Via Cinthia, 80126 Napoli, and CEINGE-Biotecnologie Avanzate S.c.ar.l., Napoli, Italy
Received 7 October 2004/ Accepted 25 February 2005
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ABSTRACT |
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INTRODUCTION |
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In P. stutzeri, (methyl)benzenes are initially activated by sequential introduction of two adjacent hydroxyl groups to form (methyl)phenols and, eventually, (methyl)catechols (Fig. 1) (5). These metabolites are subsequently cleaved into 2-hydroxymuconic semialdehyde derivatives, which, upon further processing, are transformed into citric acid cycle intermediates (1). We have recently demonstrated (8, 9) that the initial hydroxylation steps are carried out by two evolutionarily distinct bacterial multicomponent monooxygenases (BMMs), toluene o-xylene monooxygenase (ToMO), belonging to the family consisting of four-component aromatic/alkene monooxygenases (group 2 BMMs) (6, 9), and phenol hydroxylase (PH), belonging to the group consisting of toluene 2-monooxygenases (T2MO)/phenol hydroxylases (group 1 BMMs) (8). The ring cleavage step that allows aromatic hydrocarbons access to the lower metabolic pathway is catalyzed by a catechol 2,3-dioxygenase (C2,3O) (1).
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In the present work we investigated toluene and o-xylene oxidation using recombinant Escherichia coli cells expressing ToMO and PH complexes to collect data on how the biochemical properties of the two complexes could influence the efficiency of methylated aromatic utilization. Our results confirmed the general hypothesis that the two monooxygenases of P. stutzeri OX1 form a metabolic chain, thus expanding the catabolic potential of the microorganism by optimizing the degradation of benzene, toluene, and o-xylene.
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MATERIALS AND METHODS |
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Expression vectors.
Plasmids pBZ1260 (5) and pJSX148 (1) containing the ToMO and PH gene clusters, respectively, were kindly supplied by P. Barbieri (Dipartimento di Biologia Strutturale e Funzionale, Università dell'Insubria, Varese, Italy). Plasmid pBZ1260 containing the tou gene cluster cloned in the pGEM-3Z vector was used to express ToMO. For expression of PH, the DNA sequence coding for the PH complex from plasmid pJSX148 was subcloned into the vectors pGEM-3Z and pBZ1260. To do this, the DNA fragment coding for the PH cluster was excised from the pJSX148 vector using XbaI restriction endonuclease, purified by agarose gel electrophoresis, ligated with the pGEM-3Z and pBZ1260 vectors previously cut with the same enzyme, and used to transform JM101 competent cells. The resulting recombinant plasmids were designated pGEM-3Z/PH and pGEM-3Z/ToMO-PH, respectively. It should be noted that vector pGEM-3Z/ToMO-PH allows transcription of ToMO and PH open reading frames into a single mRNA under control of the lac promoter of the pGEM-3Z vector. Plasmids pGEM-3Z/PH and pGEM-3Z/ToMO-PH were used to express the PH complex alone and to coexpress the ToMO and PH complexes, respectively.
Whole-cell assays.
The assays were performed as described previously (8) using E. coli JM109 cells transformed with plasmids pGEM-3Z/PH, pBZ1260, and pGEM-3Z/ToMO-PH, which expressed PH, ToMO, and PH/ToMO complexes, respectively. The recombinant strains were routinely grown in Luria-Bertani medium (27) supplemented with 50 µg/ml ampicillin at 37°C to an optical density at 600 nm (OD600) of 
0.5. Expression of the recombinant protein was induced with 0.4 mM isopropyl-ß-D-thiogalactopyranoside at 37°C in the presence of 100 µM Fe(NH4)2(SO4)2. One hour after induction, cells were collected by centrifugation and suspended in M9 minimal medium containing 0.4% glucose. The enzymatic activity of cells was measured with phenol as the substrate by monitoring the production of catechol in continuous coupled assays with recombinant C2,3O from P. stutzeri OX1, as described previously (8). The specific activities of the cells ranged from 10 to 14 mU/OD600 for cells expressing PH and from 14 to 16 mU/OD600 for cells expressing ToMO. One milliunit was defined as the amount of catalyst that oxidized 1 nmol of phenol per min at 25°C.
Determination of apparent kinetic parameters.
All the kinetic parameters were determined using whole cells. The apparent kinetic parameters for phenol, o-, m-, and p-cresol, and 2,3-dimethylphenol (2,3-DMP) hydroxylation were determined by a continuous colorimetric assay coupled with C2,3O as described elsewhere (8). Induced cells were used at a concentration of 0.25 to 0.5 mU/ml. Substrates were added from stock solutions in water at a maximum concentration of 1 mM. The amounts of semialdehyde produced from catechol, 3-methylcatechol (3-MC), 4-MC, and 3,4-dimethylcatechol (3,4-DMC) were determined by measuring the increase in absorbance at 375 nm (
375 = 29,100 M–1 cm–1), at 388 nm (388 = 6,100 M–1 cm–1), at 382 nm (382 = 21,550 M–1 cm–1), and at 324 nm (324 = 15,310 M–1 cm–1), respectively, for the different semialdehyde products.
The apparent kinetic parameters for benzene, toluene, o-xylene, and 3,4-DMP were determined by a discontinuous assay with cells suspended at a concentration of 0.5 to 1 mU/ml in 500 µl (final volume) of M9 minimal medium containing 0.4% glucose at 25°C. Reactions were started by addition to cell suspensions of different amounts of substrates (final concentration, up to 1 mM) in N,N-dimethylformamide. The reactions were stopped at different times by addition of 50 µl of 1 M HCl. Samples were centrifuged at 12,000 rpm for 20 min at 4°C. The soluble fractions were stored at –20°C until further analysis by high-pressure liquid chromatography (HPLC) as described below.
Maximum rates (Vmax) for all substrates were always normalized to the maximum rate measured for phenol in a parallel assay. This was done for each sample of induced cells, and the experiment was repeated for each substrate with at least three independent samples of cells. For each sample of induced cells, the levels of expression of ToMO or PH were measured as described below and used to calculate the apparent kcat for phenol. The amounts of ToMO and PH complexes were determined by densitometric scanning of Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels (22) containing cell extracts. Five different amounts of samples of lysed cells were run on a single gel together with five different amounts of purified monooxygenases used as standards. ToMO and PH were expressed at similar levels.
Apparent kcat values for each substrate were calculated by determining the products of the apparent kcat for phenol and the individual substrate Vmax/phenol Vmax ratios determined as described above. Relative errors for the apparent kcat values were calculated by adding the relative error for the apparent kcat for phenol and the relative errors for the substrate Vmax/phenol Vmax ratios.
Apparent kinetic parameters were calculated with the program GraphPad Prism (GraphPad Software).
Identification of products.
Reaction products were identified with a HPLC system equipped with a Waters 1525 binary pump coupled to a Waters 2996 photodiode array detector. Mono- and dihydroxylated products were separated using an Ultrasphere C18 reverse-phase column (4.6 by 250 mm; pore size, 80 Å), and the absorbance of the eluate at 274 nm was monitored. Separation was carried out at a flow rate of 1 ml/min by using a two-solvent system comprising a 0.1% formic acid solution in water (solvent A) and a 0.1% formic acid solution in methanol (solvent B). Phenol and catechol were separated using 12 min of isocratic elution with 10% of solvent B, followed elution with a linear 10 to 30% solvent B gradient in 10 min and an isocratic 30% solvent B step. The retention times of phenol and catechol were 25.2 and 14 min, respectively. 2,3-DMP, 3,4-DMP, 3,4-DMC, and 4,5-DMC were separated by isocratic elution with 40% solvent B; the retention times for these compounds were 26.7, 24.4, 14, and 11.4 min, respectively.
Oxidation products obtained from toluene were separated using a two-solvent system comprising a 0.1% formic acid solution in water (solvent A) and a 0.1% formic acid solution in acetonitrile (solvent B). 4-Methylcatechol, 3-methylcatechol, and m-, p-, and o-cresols were separated using 5 min of isocratic elution with 15% solvent B, followed by elution with a linear 15 to 25% solvent B gradient in 5 min and then an isocratic 25% solvent B step. Under these conditions m- and p-cresols were not separated, and they eluted in a single peak at 22 min. The retention times of 4-methylcatechol, 3-methylcatechol, and o-cresol were 13.1, 14.1, and 23.3 min, respectively. The products were identified by comparing their HPLC retention times and UV-visible spectra with those of standard solutions. The amount of each product was determined by comparing the area of the peak with the areas obtained using known concentrations of standards.
The regiospecificity of toluene hydroxylation was determined by HPLC using an acetylated Cyclobond I 2000 column (Advanced Separation Technologies Inc.) and monitoring the absorbance of the eluate at 274 nm. The acetylated ß-cyclodextrin-silica column allowed separation of the three cresol isomers without extraction, which is required by the usual gas chromatography analysis (23, 32). Separation was carried out at a flow rate of 1 ml/min using a two-solvent system comprising 20 mM ammonium acetate in water (pH 5.0) (solvent A) and methanol (solvent B). o- m- and p-cresols were separated using 5 min of isocratic elution with 20% solvent B, followed by elution with a linear gradient from 20 to 60% solvent B in 20 min. The retention times of o-, m-, and p-cresols were 12.8, 14.5, and 15.2 min, respectively.
Time course of toluene and o-xylene oxidation.
The rate of product formation by E. coli cells expressing PH, ToMO, or a mixture of the two proteins was measured by the HPLC discontinuous assay. Each assay mixture contained either 30 µM toluene or 20 µM o-xylene. All cells were used at a concentration of 1 mU/ml when toluene was used as substrate and at a concentration of 1.5 mU/ml when o-xylene was used.
Rate of (di)methylcatechol production as a function of ToMO concentration.
The rate of 3- and 4-MC production from toluene was measured as a function of the ToMO concentration at ToMO concentrations ranging from 0.29 to 2.32 mU/ml with a constant concentration of cells expressing PH (0.29 mU/ml) in the presence of 30 µM toluene. The reactions in aliquots (500 µl) were stopped at 5, 10, and 15 min by adding 50 µl of 1 M HCl, and the products were analyzed by the HPLC system as described above.
The rate of 3,4-DMC production from 40 µM o-xylene was measured as a function of the ToMO concentration at ToMO concentrations ranging from 0.3 to 6 mU/ml with a constant concentration of cells expressing PH (range, 0.36 to 1 mU/ml). 3,4-DMC was measured by the continuous coupled assay with C2,3O (3 U/ml). Experimental data were fitted to the following equation: rate = (Vmax x [ToMO])/(K + [ToMO]) where Vmax is the maximum rate and K is a constant corresponding to the ToMO concentration which gives a rate of Vmax/2.
The rate of 3,4-DMC production from 40 µM o-xylene was also measured by using E. coli cells harboring vector pGEM-3Z/ToMO-PH. The cell concentration corresponded to 1.17 mU/ml of ToMO and 0.5 mU/ml of PH. An assay with phenol was used to determine the total monooxygenase activity of cells. The enzymatic activity of ToMO was measured by assaying the conversion of 3,4-DMP to 4,5-DMC because this product is exclusively formed by ToMO (see below and Fig. 1). The activity of PH was calculated by determining the difference between the total activity and the ToMO activity.
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RESULTS |
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The relative abundance of each isomer was determined by incubating E. coli cells expressing ToMO or PH with toluene or the three cresol isomers at concentrations ranging from 0.1 to 1 mM as described in Materials and Methods. The data in Table 1 indicate that p-cresol and o-cresol are the main products of toluene oxidation by ToMO and that there is slightly more of the para isomer. The hydroxylation of toluene by PH-expressing cells also resulted in a mixture of products, but in this case the ortho isomer accounted for about two-thirds of the cresol produced. Both enzymes were found to add the second hydroxyl group ortho to the hydroxyl group already present on the aromatic ring, thus producing only 3-MC from o-cresol and only 4-MC from p-cresol (data not shown). The oxidation of m-cresol catalyzed by ToMO produced almost exclusively 4-MC, whereas PH produced predominantly 3-MC (Table 1).
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Kinetic parameters of ToMO and PH with o-xylene.
The kinetic parameters of ToMO and PH with o-xylene for the first and second reaction steps were determined by whole-cell assays and are shown in Table 2. ToMO had a specificity constant for o-xylene that was 17 times higher than that of PH, whereas PH was about four times more active than ToMO with both 2,3-DMP and 3,4-DMP. In the second hydroxylation step both monooxygenases exhibited kcat/Km values for 3,4-DMP that were about 10 times higher than those measured for 2,3-DMP.
We also determined by HPLC the production of dimethylphenols and dimethylcatechols as a function of time (Fig. 4). When E. coli cells expressing ToMO (1.5 mU/ml) were incubated with 20 µM o-xylene, dimethylphenols were produced at a linear rate, whereas dimethylcatechols were detected only after about 15 min (Fig. 4A). In contrast, when E. coli cells expressing PH (1.5 mU/ml) were incubated with 20 µM o-xylene, only 2,3-DMP and 3,4-DMC were detected (Fig. 4B). The concentration of 2,3-DMP reached a low but constant value after about 30 min, whereas no 3,4-DMP was detected. 3,4-DMC accumulated at a low but constant rate after a short lag period (about 5 min). No 4,5-DMC was detected. When a mixture of E. coli cells expressing PH and ToMO (both at a concentration of 1.5 mU/ml) was incubated with 20 µM o-xylene, only 2,3-DMP and 3,4-DMC were detected (Fig. 4C), as was the case for cells expressing only PH, but 3,4-DMC was produced at a rate that was about 13 times higher.
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DISCUSSION |
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Toluene metabolism.
ToMO and PH are able to hydroxylate both toluene and cresols, confirming that the conversion of toluene into methylcatechol is a two-step process that requires cresol intermediates (Fig. 1A) (1). This is also true for the conversion of benzene to catechol by these enzymes (8, 9). The regioselectivities of ToMO and PH, however, are different. Cresols are produced by ToMO with a relaxed regioselectivity, preferentially yielding the ortho and para isomers. Such an unusual relaxed regioselectivity has been reported previously only for benzene monooxygenase from Pseudomonas aeruginosa JI104 (19), whereas most of the other characterized monooxygenases belonging to the family of four-component aromatic/alkene monooxygenases exhibit more restricted regioselectivity (15-17, 24, 34).
PH, which belongs to the large family of three-component T2MO/PHs (8), yields the three cresol isomers, but there is a preference for the ortho isomer, which accounts for 70% of the product. Thus, PH is more restricted in its regioselectivity than ToMO, but it is more relaxed than other group 1 BMMs, like T2MO, which produce only o-cresol (16, 17, 24).
It should also be noted that PH and ToMO still have opposite regioselectivities when they act on a hydroxylated substrate. For example, 4-MC is the major product of ToMO reactivity with m-cresol, whereas 3-MC is the predominant product of PH catalysis with the same substrate.
The catalytic constants of the two monooxygenases provide insight into their substrate specificities (Table 2). The specificity constant of ToMO for toluene is about 16-fold lower than that for benzene. This considerable difference is essentially due to the Km with toluene, which is about 20-fold higher than that with benzene, whereas the kcat values are very similar for these two substrates. Thus, ToMO is a more efficient catalyst for benzene oxidation. The Km of ToMO with toluene is very similar to that of toluene 4-monooxygenase (T4MO) (23); however, its kcat and kcat/Km values are sevenfold lower. These data imply that T4MO is a better catalyst for toluene transformation than ToMO. On the other hand, a different picture emerges when the data for benzene transformation are compared. T4MO has a specificity constant for benzene (25) that is lower than that previously reported for ToMO for the same substrate (8). Thus, benzene is an optimal substrate for ToMO, whereas toluene is transformed better by T4MO. This conclusion agrees with the metabolic features of Pseudomonas mendocina KR1 and P. stutzeri OX1. P. mendocina KR1 is highly specialized for growth on toluene (34), while P. stutzeri OX1, which is more versatile, can grow efficiently on both benzene and toluene (3, 5; P. Barbieri, personal communication).
When the values of the kinetic constants of PH with toluene are compared to those measured with benzene, a different picture emerges than the picture obtained for ToMO. All the kinetic constants for PH are very similar for both substrates (Table 2), suggesting that PH does not discriminate between benzene and toluene. Moreover, the specificity constant for toluene is 16-fold lower than that of ToMO, and this indicates that PH is a less efficient catalyst than ToMO with both of these hydrophobic substrates.
The specificity constants of ToMO for p- and m-cresols are identical to each other and are threefold lower than that for toluene, whereas the specificity constant for o-cresol is very similar to that for toluene. Since o-cresol accounts for only 36% of the products of the first hydroxylation reaction, we concluded that ToMO has a higher catalytic efficiency in the first step of toluene hydroxylation than in the second step, even if the difference is less pronounced than that observed in the case of benzene hydroxylation (Table 2). On the other hand, the specificity constants of PH for o- and m-cresols are similar and are about 4-5 fold higher than those for toluene. The kcat/Km value with p-cresol is about 190 times higher than that with toluene and is very similar to that with phenol. Therefore, PH shows greater catalytic efficiency in the second step of hydroxylation, and p-cresol and phenol are the preferred substrates. Taken together, these results suggest that in P. stutzeri ToMO could act as a toluene-oxidizing enzyme and PH could act as a cresol-oxidizing enzyme. The time-dependent conversion of toluene to methylcatechol reinforces this hypothesis (Fig. 2) because in the recombinant system that we used the combination of the two monooxygenases converted toluene to methylcatechol much more efficiently than ToMO or PH alone. Moreover, the percentages of 3- and 4-MC that we measured are almost identical to the values that can be calculated by assuming that ToMO catalyzes the first hydroxylation step and PH catalyzes the second hydroxylation step (see above).
o-Xylene metabolism.
A large number of bacteria are able to use m- and p-xylenes as growth substrates (2, 10, 20, 21, 36), but few of these bacteria are known to grow on o-xylene; the bacteria that can grow on o-xylene include P. stutzeri OX1 (1, 3, 5), Corynebacterium sp. strain C125 (29), and some Rhodococcus strains (7, 11, 18). Catabolism of m- and p-xylenes usually occurs through the progressive oxidation of a methyl group (2, 10), whereas o-xylene catabolism occurs by direct hydroxylation of the aromatic ring (12, 14), as is the case in P. stutzeri (1, 5).
o-Xylene hydroxylation by ToMO and PH produces 3,4- and 4,5-DMC via the intermediate formation of 2,3- and 3,4-DMP (Fig. 1B). Only 3,4-DMC, however, can be oxidized by C2,3O, the enzyme that opens the gate to the lower pathway. 4,5-DMC is not a substrate for this enzyme (1) and is therefore a dead-end product of o-xylene metabolism that results in the loss of carbon atoms from the growth substrate and in the useless depletion of NADH used by ToMO and PH in the hydroxylation reactions.
Several studies have shown that only 2,3-DMP and 3,4-DMC can be detected in the culture medium of o-xylene-oxidizing bacteria (7, 11, 18, 29), including P. stutzeri (3), for which monooxygenase-mediated o-xylene catabolism has been reported. Thus, the absence of other dimethylphenol or dimethylcatechol isomers suggests that a restricted regiospecificity of the monooxygenases acting on o-xylene would channel this substrate to the exclusive formation of 2,3-DMP in the first hydroxylation step and to 3,4-DMC in the second hydroxylation step (7, 11, 18, 29).
The data that we have collected indicate that both ToMO and PH alone are able to transform o-xylene into dimethylcatechols through a two-step process (Table 2), in which dimethylphenol intermediates are further hydroxylated to dimethylcatechols (Fig. 1A) (4). However the regioselectivities of the two enzymes are different in both reaction steps. ToMO converts o-xylene predominantly to 3,4-DMP, whereas PH produces predominantly 2,3-DMP (Table 1). Both monooxygenases convert 2,3-DMP to 3,4-DMC, but they exhibit different regioselectivities with 3,4-DMP. ToMO oxidizes 3,4-DMP mainly to 4,5-DMC, which cannot be further metabolized (5). PH converts 3,4-DMP exclusively to 3,4-DMC. Thus, our data indicate that only PH can produce a dimethylcatechol which can be further metabolized through the lower pathway.
The catalytic constants of the two enzymes reported in Table 2 clearly indicate that ToMO is a more efficient catalyst than PH in the first hydroxylation step and has a 17.7-fold-higher specificity constant. On the other hand, PH is more efficient than ToMO in the second hydroxylation step and has specificity constants for 2,3- and 3,4-DMP that are about three- and fourfold higher, respectively, than those determined for ToMO. Our kinetic data indicate that 3,4-DMP is transformed into products more efficiently than 2,3-DMP, especially by PH, which has a kcat/Km that is fivefold higher than that of ToMO. The regioselectivity and the kinetic constants of ToMO are in agreement with the finding that when E. coli cells expressing ToMO are incubated with o-xylene, only 4,5-DMC is produced after a long lag phase (Fig. 4A). On the other hand, E. coli cells expressing PH produce 3,4-DMC, but at a very low rate, likely because of the poor conversion of o-xylene to dimethylphenol in the first step (Fig. 4B).
A mixture of E. coli cells expressing PH and ToMO produced only 2,3-DMP and 3,4-DMC (Fig. 4C). However, in this case, no lag phase was observed in the production of 3,4-DMC, and the rate of 3,4-DMC formation was 13-fold greater than the rate recorded with cells expressing PH alone. 2,3-DMP, on the other hand, was produced at a low and constant level (Fig. 4C). The fact that 4,5-DMC was not detected in this mixture is consistent with the conclusion that the second hydroxylation step is performed by PH. 2,3-DMP accumulated in these reactions because it is not a good substrate either for PH or for ToMO, whereas 3,4-DMP was not detected likely because it is the preferred substrate for PH.
The rate of 3,4-DMC formation catalyzed by mixtures of different amounts of cells expressing ToMO and a constant amount of cells expressing PH was found to be proportional to the concentration of ToMO (Fig. 5), and there was a hyperbolic dependence on the ToMO concentration. According to the conclusion described above that ToMO catalyzes the first step of the reaction by producing dimethyphenols, which in turn are hydroxylated to 3,4-DMC by PH, low concentrations of ToMO would be limiting for the system; hence, the rate of 3,4-DMC formation would increase when the ToMO concentration increased. On the other hand, higher ToMO concentrations would provide PH with a saturating amount of DMP isomers. Under these conditions, the PH concentration would be the rate-limiting factor for production of 3,4-DMC, whose rate of formation should be independent of the ToMO concentration. This behavior is exactly what was experimentally observed (Fig. 5). Moreover, when ToMO was used at a constant concentration, the maximum rate of 3,4-dimethylcatechol formation increased linearly with increasing PH concentrations (Fig. 5, inset).
The rate of 3,4-DMC formation was also measured using E. coli cells coexpressing ToMO and PH and was found to be almost identical to the rate measured using the same amounts of ToMO and PH expressed in different cells (Fig. 5). Therefore, the presence of ToMO and PH in different cellular compartments does not influence either the percentages of the products of the conversion of o-xylene to dimethylcatechols or their rate of formation.
In conclusion, the data for the oxidation of toluene and o-xylene, together with the data reported previously for the transformation of benzene (8), strongly support a general hypothesis concerning the metabolism of aromatic compounds in P. stutzeri OX1. ToMO and PH act sequentially and allow efficient conversion of nonhydroxylated aromatic hydrocarbons to substituted catechols. Moreover, this coupling is particularly important in the case of o-xylene degradation because the sequential action leads to production of 3,4-DMC, the sole dimethylcatechol that can enter the lower pathway and lead to the production of precursors for biosynthetic reactions. This hypothesis also provides a metabolic explanation for the acquisition of the ToMO operon by P. stutzeri OX1 (4). The use of the two enzymes in a concerted fashion confers on the strain a selective advantage based on the ability to maximize the efficiency of exploitation of nonhydroxylated aromatic hydrocarbons, such as benzene, toluene, and o-xylene.
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ACKNOWLEDGMENTS |
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This work was supported by grants PRIN/2000 and PRIN/2002 from the Ministry of University and Research.
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FOOTNOTES |
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V.C. and E.N. contributed equally to this paper.
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REFERENCES |
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), m- and p-cresols (
), 3-methylcatechol (
), and 4-methylcatechol (•) by cells expressing ToMO at a concentration of 1 mU/ml (A), cells expressing PH at a concentration of 1 mU/ml (B), and a mixture of cells expressing ToMO (1 mU/ml) and cells expressing PH (1 mU/ml) (C) incubated with 30 µM toluene.
) in the presence of 40 µM o-xylene. The solid circle indicates the rate of 3,4-DMC production by cells coexpressing ToMO (1.17 mU/ml) and PH (0.5 mU/ml). The rate of 3,4-DMC production was measured by the continuous coupled assay with C2,3O as described in Materials and Methods. The inset shows the rate of 3,4-DMC formation as a function of the PH concentration at a constant concentration of ToMO (4.5 mU/ml).