(MhpB) and Alcaligenes eutrophus (MpcI) - Journal of Bacteriology

0 downloads 0 Views 359KB Size Report
Copyright. 1996, American Society for Microbiology .... methanol-H2SO4 and was recrystallized from diethyl ether-light petroleum: melt- ing point 86 to ...... University Science Books, Mill. Valley, Calif. ... laboratory manual, 2nd ed. Cold Spring ...
JOURNAL OF BACTERIOLOGY, Sept. 1996, p. 5249–5256 0021-9193/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 178, No. 17

Catechol Dioxygenases from Escherichia coli (MhpB) and Alcaligenes eutrophus (MpcI): Sequence Analysis and Biochemical Properties of a Third Family of Extradiol Dioxygenases EMMA L. SPENCE,1 MAKOTO KAWAMUKAI,2 JONATHAN SANVOISIN,1 HELEN BRAVEN,1 1 AND TIMOTHY D. H. BUGG * Department of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, United Kingdom,1 and Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690, Japan2 Received 29 April 1996/Accepted 18 June 1996

The nucleotide sequence of the Escherichia coli mhpB gene, encoding 2,3-dihydroxyphenylpropionate 1,2dioxygenase, was determined by sequencing of a 3.1-kb fragment of DNA from Kohara phage 139. The inferred amino acid sequence showed 58% sequence identity with the sequence of an extradiol dioxygenase, MpcI, from Alcaligenes eutrophus and 10 to 20% sequence identity with protocatechuate 4,5-dioxygenase from Pseudomonas paucimobilis, with 3,4-dihydroxyphenylacetate 2,3-dioxygenase from E. coli, and with human 3-hydroxyanthranilate dioxygenase. Sequence similarity between the N- and C-terminal halves of this new family of dioxygenases was detected, with conserved histidine residues in the N-terminal domain. A model is proposed to account for the relationship between this family of enzymes and other extradiol dioxygenases. The A. eutrophus MpcI enzyme was expressed in E. coli, purified, and characterized as a protein with a subunit size of 33.8 kDa. Purified MhpB and MpcI showed similar substrate specificities for a range of 3-substituted catechols, and evidence for essential histidine and cysteine residues in both enzymes was obtained. (6). The mhpB gene, encoding the dioxygenase enzyme responsible for this cleavage, has been mapped to min 8 of the E. coli chromosome (5) and has been subcloned and overexpressed (4). The MhpB gene product has been purified to homogeneity and was found to be dependent upon iron(II) for activity. Peptide sequencing of the N-terminal 15 amino acids of the purified protein revealed sequence similarity with the inferred sequence of the dioxygenase MpcI from A. eutrophus (4). The apparent sequence similarity of the two enzymes prompted us to investigate both the nucleotide sequence of the E. coli mhpB gene and the biochemical properties of the A. eutrophus enzyme. Here we report the identification from these studies of a distinct family of extradiol dioxygenases.

The non-heme iron-dependent catechol dioxygenases catalyze the oxidative cleavage of catechol intermediates in a number of bacterial pathways for the degradation of aromatic hydrocarbons (11). These pathways are to a large extent responsible for the bacterial degradation of naturally occurring and man-made aromatic chemicals in the environment. Nucleotide sequencing of a number of genes encoding catechol dioxygenases has revealed that the iron(III)-dependent intradiol dioxygenases form one ancestral family of enzymes, while most of the iron(II)-dependent extradiol dioxygenases fall into a separate family (11, 12). However, a small number of extradiol dioxygenases appear to show no sequence similarity with the major family of enzymes, namely, protocatechuate 4,5-dioxygenase from Pseudomonas paucimobilis (17), 3,4-dihydroxyphenylacetate 2,3-dioxygenase from Escherichia coli (19), and an extradiol dioxygenase, MpcI, from Alcaligenes eutrophus (14). Furthermore, three much smaller dioxygenase enzymes (21 kDa) have been identified in a naphthalene sulfonate-degrading strain, BN6 (13), and a polychlorinated biphenyl-degrading Rhodococcus, strain P6 (1); these are also distinct from the major family of enzymes. The recently obtained X-ray crystal structure of 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas strain LB400 (10) has revealed that this extradiol dioxygenase contains two domains with very similar tertiary structures, although only the C-terminal domain binds an iron(II) cofactor. The sequence of the C-terminal domain can be aligned with that of the small Rhodococcus strain P6 dioxygenase, suggesting that this type of enzyme has evolved from a gene duplication event (10). A pathway responsible for the degradation of 3-phenylpropionic acid has been identified in E. coli; this pathway proceeds via the extradiol cleavage of 2,3-dihydroxyphenylpropionic acid

MATERIALS AND METHODS Materials. Catecholic substrates were either synthesized or purchased from Aldrich Chemical Co. 2,3-Dihydroxyphenylpropionic acid was prepared by the method of Blakley and Simpson (2). 2,3-Dihydroxyphenoxyacetic acid was prepared by the method of Christiansen (8). 2,3-Dihydroxycinnamic acid was prepared by demethylation of 2,3-dimethoxycinnamic acid (Aldrich), as described elsewhere (23). Methyl 2,3-dihydroxyphenylpropionate was prepared in 89% yield by acid-catalyzed esterification (24) of 2,3-dihydroxyphenylpropionic acid in methanol-H2SO4 and was recrystallized from diethyl ether-light petroleum: melting point 86 to 878C; infrared (IR) (solution) 3422, 1711, 1621, 1595 cm21; 1H nuclear magnetic resonance (NMR) (300 MHz, CDCl3) dH 6.78 (2H, m), 6.62 (1H, dd, J 5 6.2, 2.9 Hz), 3.71 (3H, s), 2.90 (2H, t, J 5 7 Hz), 2.75 (2H, t, J 5 7 Hz) ppm; m/z 196 (M1). E. coli JM109/pAE166 containing the A. eutrophus mpcI gene was kindly provided by P. Fortnagel (University of Hamburg). Preparation of 3-substituted catechols. The synthetic scheme shown in Fig. 1 was employed. (i) Preparation of compounds 1a to 1c. Sodium hydride (60% dispersion in mineral oil, 2.85 g, 71 mmol) was added to dry degassed dimethyl sulfoxide (100 ml), and the resultant mixture was heated at 758C until a homogeneous solution was obtained. The solution was cooled to room temperature, methyltriphenylphosphonium bromide (25.0 g, 70 mmol) was added in portions, and the mixture was stirred for a further 45 min. Addition of 2,3-dimethoxybenzaldehyde (8.0 g, 48 mmol) resulted in an exothermic reaction, and the reaction mixture was stirred at 508C for 1 h. After cooling, the mixture was taken up in water (250 ml) and extracted into ether (three times, 100 ml each). Removal of the solvent at reduced pressure gave a crude brown solid, which was distilled under reduced

* Corresponding author. Phone: 01703-593816. Fax: 01703-593781. 5249

5250

SPENCE ET AL.

J. BACTERIOL.

FIG. 1. Synthetic route used for the preparation of 3-ethyl-, 3-propyl-, and 3-phenethylcatechols.

pressure to give compound 1a as a colorless oil (4.22 g, 54%): boiling point 1188C (5 mm Hg); IR (liquid film) 1629, 1575 cm21; 1H NMR (300 MHz, CDCl3) dH 7.14 (2H, m), 7.04 (1H, t, J 5 8 Hz), 6.85 (1H, dd, J 5 8, 1.1 Hz), 5.79 (1H, dd, J 5 17.6, 1.1 Hz), 5.34 (1H, dd, J 5 11.0, 1.1 Hz), 3.88 (3H, s), 3.85 (3H, s) ppm; m/z 164 (M1, 100%), 149 (55%), 121 (65%). Compound 1b was made by the same method in 61% yield, using ethyltriphenylphosphonium bromide. The product was isolated as a 2:1 mixture of Z and E alkenes, respectively: boiling point 38 to 488C (0.03 mm Hg); IR (liquid film) 2834, 1654, 1575 cm21; 1H NMR (300 MHz, CDCl3) dH Z alkene 1.84 (3H, d, J 5 9 Hz), 3.75 (3H, s), 3.86 (3H, s), 5.85 (1H, dq, J 5 12,9 Hz), 6.57 (1H, d, J 5 12 Hz), 6.8 to 7.0 (3H, m); E alkene 1.90 (3H, d, J 5 9 Hz), 3.76 (3H, s), 3.85 (3H, s), 6.25 (1H, dq, J 5 17,9 Hz), 6.70 (1H, d, J 5 17 Hz), 6.8 to 7.0 (3H, m) ppm. Compound 1c was made by the same method in 63% yield, using benzyltriphenylphosphonium bromide: IR (liquid film) 2833, 1576 cm21; 1H NMR (60 MHz, CDCl3) dH 3.70 (3H, s), 3.75 (3H, s), 6.5 to 6.8 (5H, m), 7.0 to 7.5 (6H, m) ppm. (ii) Preparation of compounds 2a to 2c. To a degassed solution of compound 1a (1.0 g, 6.1 mmol) in ethanol (50 ml) was added 10% palladium on charcoal (100 mg), and the mixture was stirred overnight under an atmosphere of hydrogen. The reaction mixture was filtered, and the solvent was removed at reduced pressure to give 1,2-dimethoxy-3-ethylbenzene as a colorless oil (0.68 g, 67%). This oil was dissolved in dichloromethane (10 ml) and was added to a solution of boron tribromide (2.3 g, 9.0 mmol) in dry dichloromethane (5 ml) at 2788C under nitrogen. The mixture was allowed to warm to room temperature and was stirred for a further 24 h. Water (30 ml) was carefully added, and the product was extracted into ether (six times, 20 ml each). The extracts were washed with water (three times, 20 ml each) and dried (MgSO4), and the solvent was removed under reduced pressure to yield the product as a brown viscous oil. Purification was carried out by silica column chromatography, with elution with ethyl acetate-light petroleum (1:1), yielding compound 2a as a brown solid (0.49 g, 58% overall): melting point 67 to 698C (literature, 70 to 728C [18]); IR (liquid film) 3410, 1621, 1594 cm21; 1H NMR (300 MHz, CDCl3) dH 6.74 (3H, m), 2.66 (2H, q, J 5 7.5 Hz), 1.24 (3H, t, J 5 7.5 Hz) ppm; m/z 138 (M1). Compound 2b was synthesized in 60% yield by the above-described method: IR (Nujol mull) 3355, 1591 cm21; 1H NMR (60 MHz, CDCl3) dH 1.0 (3H, t, J 5 7 Hz), 1.7 (2H, sextet, J 5 7 Hz), 2.65 (2H, t, J 5 7 Hz), 7.15 (1H, t, J 5 8 Hz), 7.25 (1H, d, J 5 8 Hz), 7.52 (1H, d, J 5 8 Hz) ppm. Compound 2c was synthesized in 55% yield by the above-described method: IR (Nujol mull) 3459, 3341, 1624, 1586 cm21; 1H NMR (60 MHz, CDCl3) dH 3.0 (4H, s), 7.15 to 7.4 (7H, br s), 7.6 (1H, d, J 5 8 Hz) ppm. Nucleotide sequencing of the mhpB gene. Kohara phage 139 was propagated in E. coli LE392, and the lambda DNA was purified by a standard protocol (20). A 3.1-kb SmaI-PstI DNA fragment was excised and was subcloned into the plasmids pBluescript KS1 and pBluescript KS2. A sequence deletion set for the 3.1-kb cloned region was constructed by using a Takara deletion kit for kilobase sequencing, according to the manufacturer’s instructions. Deletion clones were made in both directions. DNA sequencing was carried out by the dideoxy-chain termination method (21) with a Pharmacia ALF II DNA sequencer. Enzyme assays. MhpB and MpcI were routinely assayed by monitoring the increase in A394 upon addition of the enzyme to 1 mM 2,3-dihydroxyphenylpropionic acid in 50 mM Tris (pH 8.0) (ε394 5 19,150 M21 cm21 for ring fission product) at 208C. In crude extracts MpcI was assayed without reactivation, but after ammonium sulfate precipitation the enzyme was reactivated as required. Reactivation was carried out by incubation of freshly prepared ammonium iron(II) sulfate (5 mM) and sodium ascorbate (5 mM) with enzyme solutions for 5 min at 08C. Relative rates for alternative substrates were determined by using a Clark-type oxygen electrode, using 10 mM substrate in 50 mM Tris (pH 8.0). In selected cases Km values were determined by UV spectroscopy. Rates of product formation were determined at a range of substrate concentrations (0.2 to 5 Km), at the lmax of the corresponding ring fission product. Km values were determined from triplicate rate determinations by using Lineweaver-Burk or Eadie-Hofstee plots.

Purification of A. eutrophus dioxygenase MpcI. All steps were carried out at 48C unless otherwise stated. PEG buffer consists of a degassed stock of 50 mM potassium phosphate buffer (pH 7.0) containing 10% ethanol and 10% glycerol. Suspension buffer A consists of PEG buffer to which were added 0.5 mM b-mercaptoethanol and 0.5 mM ammonium iron(II) sulfate immediately before use. Purification buffer B consists of PEG buffer to which was added 0.5 mM b-mercaptoethanol immediately before use. A 1-liter culture of JM109/pAE166 in Luria broth containing 100 mg of ampicillin per ml was grown at 308C to an A600 of 0.6, at which point 0.5 mM IPTG (isopropyl-b-D-thiogalactopyranoside) was added and growth was continued for a further 4 to 5 h at 308C. The cells were harvested by centrifugation for 10 min at 5,000 3 g and resuspended in buffer A (35 ml). Cell lysis was carried out by passage through a pneumatic cell disrupter (5 lb/in2). Cell debris was removed by centrifugation for 25 min at 25,000 3 g. Powdered ammonium sulfate was added to the crude extract to 45% saturation (267 g/liter), and the suspension was stirred for 1 h and centrifuged for 10 min at 25,000 3 g. The supernatant was loaded onto a phenyl agarose column (5.0 by 1.0 cm) and eluted with a gradient (200 ml) of buffer B containing decreasing ammonium sulfate concentrations from 1.0 to 0.0 M. Fractions containing MpcI activity, eluting at 0.9 to 0.7 M, were pooled and stored at 48C. E. coli MhpB was purified as previously described (4). Protein concentrations were measured by the method of Bradford (3), with bovine serum albumin as a standard. Purification was monitored by sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis (SDS–12% PAGE) with a Bio-Rad Mini-PROTEAN II cell, according to the manufacturer’s instructions. Chemical modification studies. Freshly prepared solutions of sodium iodoacetate, dithionitrobenzoic acid (DTNB), succinic anhydride, p-hydroxymercuribenzoate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide methiodide (EDC), and diethyl pyrocarbonate in water or ethanol were added to reactivated MhpB (0.8 U) or reactivated MpcI (0.6 U) to a final concentration of 5 to 10 mM. The incubation mixtures were stored over ice, and aliquots were removed and assayed for activity at various times by the continuous UV assay. In the case of EDC, 10 mM ethylene diamine was included in the incubation mixture. MhpB (0.7 U) and MpcI (0.6 U) were also treated with diethyl pyrocarbonate, p-hydroxymercuribenzoate, and DTNB for 10 min at 08C prior to reactivation with ammonium iron(II) sulfate (5 mM) and sodium ascorbate (5 mM). Nucleotide sequence accession number. The nucleotide sequence of the E. coli mhpB gene was deposited in GenBank and assigned accession number D86239.

RESULTS Nucleotide sequencing of the E. coli mhpB gene. The mhpB gene had previously been mapped to min 8 of the E. coli chromosome, between the lacZ and hemB genes (5), and had been subcloned from Clarke-Carbon plasmid pLC20-30 onto a 5.5-kb ClaI-HindIII DNA fragment (4). This region of DNA is known to be located on the phage 139 of the Kohara set of fragments of E. coli DNA (15). Accordingly, a 3.1-kb SmaI-PstI DNA fragment was excised from phage 139 and cloned into a pBluescript vector. A set of overlapping deletion constructs was made, and the DNA sequence was determined. Identification of the mhpB open reading frame was possible by using the previously determined protein N-terminal sequence (4), which matched the inferred amino acid sequence exactly. Translation of the mhpB open reading frame (Fig. 2) gives the inferred amino acid sequence for the mhpB gene product

VOL. 178, 1996

FAMILY OF EXTRADIOL CATECHOL DIOXYGENASES

5251

FIG. 2. Nucleotide sequence of the E. coli mhpB gene and inferred amino acid sequence of the encoded protein.

2,3-dihydroxyphenylpropionate 1,2-dioxygenase. The inferred sequence comprises 314 amino acids and gives a predicted subunit molecular mass of 34,170 Da, comparable with the value of 36 6 1 kDa estimated from SDS-PAGE of the purified enzyme (4). Amino acid sequence comparisons. The inferred MhpB amino acid sequence was found to have 58% sequence identity with the inferred sequence of the A. eutrophus MpcI dioxygenase, consistent with the similarity at the N terminus noted earlier (4). Upon closer inspection, sequence similarity between the N-terminal and C-terminal halves of both proteins was observed. An alignment of the N- and C-terminal domains of both the MhpB and MpcI sequences is shown in Fig. 3. There are 39 positions in the alignment (23% of the total) in which either identical or functional conservation is observed in

all four sequences, and there are 66 positions (39% of the total) in which there are pairwise similarities between the Nand C-terminal domains of either MhpB or MpcI. These observations suggest that these enzymes, like the Pseudomonas strain LB400 2,3-dihydroxybiphenyl 1,2-dioxygenase (10), have evolved by gene duplication at an early evolutionary point. Alignments of the MhpB amino acid sequence with those of other extradiol dioxygenases revealed 20% sequence identity with protocatechuate 4,5-dioxygenase (LigB) from P. paucimobilis (17), 12% sequence identity with 3,4-dihydroxyphenylacetate 2,3-dioxygenase (HpcB) from E. coli (18), and 10% identity with human 3-hydroxyanthranilate dioxygenase (HAO), a rare example of a mammalian extradiol dioxygenase (16). A multiple alignment of these sequences is shown in Fig. 4. It can be seen that there are several regions with a high level of

FIG. 3. Amino acid sequence alignment of the N-terminal domains (positions 1 to 171) and C-terminal domains (position 172 to end) of E. coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) and A. eutrophus catechol 2,3-dioxygenase (MpcI). #, identical amino acid in all four sequences; 1, functionally similar amino acid in all four sequences (I/L/V/M, S/T, D/E, D/N, E/Q, A/G, A/S, R/K/H, and F/Y allowed); ., pairwise similarity between N- and C-terminal domains in either MhpB or MpcI; *, positions marked in Fig. 4 and 6 corresponding to active-site residues in Pseudomonas strain LB400 dioxygenase BphC (10).

5252

SPENCE ET AL.

J. BACTERIOL.

FIG. 4. Alignment of E. coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) amino acid sequence with sequences of other extradiol catechol dioxygenases: MpcI, A. eutrophus catechol 2,3-dioxygenase (14); LigB, P. paucimobilis protocatechuate 4,5-dioxygenase (17); HpcB, E. coli 3,4-dihydroxyphenylacetate 2,3-dioxygenase (19); HAO, human HAO (16). #, identical amino acid residue in all five sequences; 1, functionally similar amino acid residue in four or five sequences. Below these sequences are aligned the amino acid sequences of the smaller extradiol dioxygenases BphCBN6 from strain BN6 (13) and BphC2P6 and BphC3P6 from Rhodococcus globerulus P6 (1). *, positions marked in Fig. 6 corresponding to active-site residues identified in Pseudomonas strain LB400 dioxygenase BphC by X-ray crystallography (10).

sequence conservation among all sequences, notably in the vicinity of His-10, His-53, His-115, and His-179 (positions in the MhpB sequence), which are conserved in all sequences, with the exception of His-115, which is not conserved in the human enzyme. Since it is known that in both intradiol and extradiol enzymes the non-heme iron cofactor is coordinated by histidine ligands (10, 11), the observation of strongly conserved histidine residues is noteworthy. Since the levels of sequence identity in the cases of LigB, HpcB, and HAO are not high, the statistical significance of the alignments was assessed by a “jumble test” (9). The LigB, HpcB, and HAO sequences were each jumbled into 500 random sequences, and each random sequence was compared with the MhpB sequence; the distribution of alignment scores was then compared with the score for alignment of the original sequence. By this analysis, the alignment score for MhpB versus LigB was 11 standard deviations above the mean score, and that for MhpB versus HpcB was 3.3 standard deviations above, indicating that the observed sequence similarity is statistically significant. The score for MhpB versus HAO was less than 1 standard deviation above the mean score; thus, the sequence similarity with HAO is not statistically significant in a pairwise comparison. However, when the HAO sequence is compared with the other four sequences, there are identical matches with one or more sequences in 74 positions (26% of the total). Furthermore, the HAO sequence contains three of the four conserved histidine residues found in this family, and it shows no sequence similarity with the major family of extradiol dioxygenases. Thus, we suggest that it be tentatively included in this family of sequences. The significance of the conserved histidine residues is confirmed by a further alignment with the sequences of the 21-kDa extradiol dioxygenases from strain BN6 (13) and from Rhodococcus strain P6 (1), as shown in Fig. 4. Although the overall

level of sequence similarity is low (12% sequence identity between the MhpB and BN6 sequences), the conserved residues His-10, His-53, and His-115 can be aligned with conserved histidine residues in the latter enzymes. Thus, there appears to be a common pattern of conserved histidine residues in both families of enzymes (see Discussion). Purification of A. eutrophus dioxygenase MpcI. The A. eutrophus mpcI gene has previously been cloned onto an expression vector, pAE166, and expressed in crude extracts of E. coli JM109 (14). In crude extracts of JM109/pAE166, the dioxygenase activity was unstable; however, we found that the activity could be substantially stabilized by addition of 10% ethanol, 10% glycerol, and 0.5 mM ammonium iron(II) sulfate to the purification buffer. Upon precipitation, chromatography, or dilution into iron(II)-free buffer, enzyme activity was rapidly lost, but it could be regained quantitatively upon reactivation with 2 mM ammonium iron(II) sulfate and 2 mM sodium ascorbate. Similar reactivation behavior has previously been observed for E. coli MhpB (4) and for other extradiol dioxygenases (7, 13). It was found that the specific activity of MpcI dropped upon passage through adsorptive chromatography media; however, purification was pursued in order to obtain enzyme free of possible contaminants for comparative studies. The dioxygenase activity was purified by a 45% ammonium sulfate precipitation followed by passage through a phenyl agarose column with a 1 to 0 M gradient of ammonium sulfate. Although the specific enzyme activity had dropped from 10.7 U/mg in the crude extract to 4.3 U/mg after phenyl agarose chromatography (Table 1), analysis by SDS-PAGE revealed only one major protein band at 34 kDa (data not shown). Attempts to purify the enzyme further to homogeneity led to an irreversible loss of activity. The molecular mass of the purified protein was estimated by SDS-PAGE to be 33.8 6 1 kDa, compared with the predicted molecular mass of 33.1 kDa (14).

VOL. 178, 1996

FAMILY OF EXTRADIOL CATECHOL DIOXYGENASES

5253

TABLE 1. Purification of A. eutrophus dioxygenase MpcIa Step

Vol (ml)

Activity (U/ml)

Protein (mg/ml)

Sp act (U/mg)

Crude extract 45% (NH4)2SO4 supernatant Phenyl agarose pool

44 44 50

126 60.9 5.8

11.8 8.8 1.35

10.7 6.9 4.3

a

Purification was carried out as described in Materials and Methods. One unit of enzyme activity is the activity required to produce 1 mmol of product per min.

Properties of dioxygenase MpcI. The purified enzyme was entirely dependent upon iron(II) for catalytic activity. Treatment with 5 mM EDTA resulted in complete loss of activity. Treatment with 5 mM sodium ascorbate or 5 mM sodium dithionite gave no loss of activity, while treatment with 5 mM hydrogen peroxide resulted in complete loss of activity. Following inactivation with EDTA, activity could be restored by addition of excess Fe21 but not with K1, Na1, Mg21, Zn21, Cu21, Mo21, Co21, or Fe31 solutions. The reactivation of MpcI by iron(II) and ascorbate was found to be time dependent, with maximum activity being obtained after 5 to 40 min. At a fixed concentration of 5 mM sodium ascorbate, the optimum iron(II) concentration was found to be 1 to 6 mM. When the reactivated MpcI was diluted 10-fold into iron(II)-free PEG buffer, a threefold loss of activity was observed after 5 min. This loss of activity upon dilution is even more marked for MhpB (half-life, 30 s) and implies that both enzymes bind the iron(II) cofactor fairly weakly (4). Substrate specificities of MhpB and MpcI. In order to assess the importance of the carboxylate group for substrate recognition, the methyl ester of 2,3-dihydroxyphenylpropionic acid was synthesized by esterification in methanol-H2SO4. The effect of removing the carboxylate group was examined by synthesizing 3-ethylcatechol and 3-propylcatechol. These compounds were synthesized by Wittig reactions of 2,3dimethoxybenzaldehyde followed by hydrogenation with 10% Pd-C and demethylation with BBr3. A 3-phenethylcatechol analog was also synthesized by the same route, which is shown in Fig. 1. A range of catechol substrates was tested as substrates with purified A. eutrophus MpcI and E. coli MhpB, and the relative rates measured by oxygen electrode assays are shown in Table 2. Michaelis-Menten kinetic behavior was observed for most

substrates, and Km values for selected substrates were measured by the continuous UV assay. The two enzymes show very similar substrate specificity profiles, accepting a range of 3-substituted catechols as substrates with comparable rates. The optimum substrate for both enzymes is 2,3-dihydroxyphenylpropionate, with a Km of 26 mM for MhpB and 7 mM for MpcI, implying a close functional similarity between the two enzymes. The methyl ester of the natural substrate was processed at almost identical rates, implying that the carboxylate group is not bound through an electrostatic interaction at the respective active sites. Similarly, both 3-ethylcatechol and 3-propylcatechol were processed efficiently by both enzymes. Surprisingly, both enzymes processed the 3-phenethylcatechol substrate, indicating that a sizeable binding site for the side chain is available. The importance of the catecholic hydroxyl groups was confirmed by the observation that neither 2,3-dimethoxyphenylpropionic acid nor 2-aminophenol was accepted as a substrate. 2,3-Dihydroxycinnamic acid was accepted as a good substrate by both enzymes, indicating that the enzyme is able to bind the alkyl side chain in a transoid conformation. A Km value of 36 mM was measured for MhpB; however, unusual kinetic behavior was observed with MpcI, as shown in Fig. 5. At high substrate concentrations substrate inhibition was observed, which has also been observed for other catechol dioxygenases (13). However, at low substrate concentrations sigmoidal kinetics was observed, suggesting cooperativity between protein subunits. Analysis with a Hill plot reveals positivenegative-positive cooperativity. Although the subunit structure of MpcI is not known (since the enzyme loses all activity upon gel filtration), it is known that MhpB exists as a tetramer (4). For a four-site enzyme the observed data could be rationalized if the binding of the second substrate shows positive cooper-

TABLE 2. Substrate specificities of dioxygenases MhpB and MpcI Substratea

Catechol 3-Methylcatechol 4-Methylcatechol 3-Ethylcatechol (2a) 3-Propylcatechol (2b) 3-Phenethylcatechol (2c) 2,3-Dihydroxyphenylpropionic acid 2,3-Dihydroxycinnamic acid 2,3-Dihydroxyphenoxyacetic acid Methyl-2,3-dihydroxyphenylpropionate

Side chain

H 3-CH3 4-CH3 3-CH2CH3 3-CH2CH2CH3 3-CH2CH2Ph 3-CH2CH2CO2H 3-CHACHCO2H 3-OCH2CO2H 3-CH2CH2CO2CH3

lmaxb (nm)

375 385 379 389 390 392 394 454 350 394

MhpB Vrelc (%)

64 58 35 71 85 23 100 99 44 89

MpcI Kmd (mM) e

700 90e ND 185 154 94 26e 36 300 37

Vrel (%)

Km (mM)

85 65 68 89 93 37 100 95 29 97

NDf 130 ND 59 ND ND 7.1 14g ND ND

a No activity was observed for 2,3-dimethoxypropionic acid, 2-aminophenol, 2,3-dihydroxybenzoic acid (3-CO2H), 3,4-dihydroxybenzoic acid (4-CO2H), 3,4dihydroxycinnamic acid (4-CHACHCO2H), 1,2,3-trihydroxybenzene (3-OH), 1,2,4-trihydroxybenzene (4-OH), or 2,3-dihydroxybenzaldehyde (3-CHO). b lmax for the ring fission product of the substrate. c Relative rate measured at a substrate concentration of 10 mM with the oxygen electrode assay. d Determined with the continuous UV assay, as described in Materials and Methods. e As determined previously (4). f ND, not determined. g Sigmoidal kinetics was observed (Fig. 5). Km estimated from log (V/Vmax 2 V) 5 0.

5254

SPENCE ET AL.

J. BACTERIOL.

FIG. 5. Kinetic behavior observed for dioxygenases MhpB and MpcI with alternate substrate 2,3-dihydroxycinnamic acid (side chain, 3-CHACHCO2H). (A) Plot of rate (relative to rate at [S] 5 10 mM) versus substrate (S) concentration, showing substrate inhibition of MpcI at [S] . 100 mM. (B) Allosteric behavior of MpcI at low substrate concentrations. A plot of log(V/Vmax 2 V) (22), which in the absence of allosteric behavior would give a straight line with a gradient of 1.0, as observed with MhpB, is shown. MpcI shows negative-positive-negative cooperativity at concentrations up to 100 mM, and substrate inhibition is apparent at higher concentrations.

ativity, binding of the third substrate shows negative cooperativity, and binding of the fourth substrate shows positive cooperativity (22). It therefore appears that for this particular substrate, a complex pattern of cooperativity between the enzyme subunits is taking place. Chemical modification of MhpB and MpcI. Evidence for essential active-site residues in dioxygenases MhpB and MpcI was sought by using chemical modification studies. The results of treatment with a range of group-specific reagents are shown in Table 3. Similar behavior is observed for the two enzymes. Both enzymes are substantially inactivated by treatment with the histidine-directed reagent diethyl pyrocarbonate. This reagent has been used previously to modify histidine residues of catechol 2,3-dioxygenase from Rhodococcus rhodochrous CTM (7). Since the amino acid sequence alignments described above have indicated the presence of conserved histidine residues, it seems likely that there are essential active-site histidine residues which may function as iron(II) ligands. Both enzymes are rapidly inactivated by the cysteine-directed reagents p-hydroxymercuribenzoate and DTNB, implying that there is a cysteine residue present at or near the active sites of both enzymes. There are two cysteine residues which

are conserved in the MhpB and MpcI sequences: Cys-7 and Cys-66. Neither residue is conserved in the sequences of other enzymes in this family; thus, a role for cysteine as an iron(II) ligand seems unlikely. However, Cys-7 is situated close in the sequence to the highly conserved His-10. Hence, it is plausible that modification of Cys-7 by a bulky reagent might block the active sites of these enzymes, leading to inactivation. This hypothesis is consistent with the observation that neither enzyme is inactivated by sodium iodoacetate, a cysteine-modifying reagent of smaller size. There is no inactivation by the carboxyldirected reagent EDC; thus, there is no evidence for essential aspartate or glutamate residues. The three reagents which demonstrated inactivation were incubated with the apoenzyme for 10 min prior to reactivation. Similar levels of inactivation were observed in each case. DISCUSSION Previous studies of E. coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) have revealed that this enzyme has sequence similarity with A. eutrophus MpcI at the N terminus of the protein sequence (4). Sequencing of the E. coli mhpB

TABLE 3. Chemical modification of MhpB and MpcIa % MhpB activity after treatment for:

% MpcI activity after treatment for:

Reagent 1 min

Sodium iodoacetate Succinic anhydride EDC p-Hydroxymercuribenzoate DTNB Diethyl pyrocarbonate a b

80 113 84 34 88 77

10 min

86 95 71 1.6 32 7.4

Assays were carried out as described in Materials and Methods. ND, not determined.

10 min before reactivation b

ND ND ND 15 3.5 6.1

1 min

10 min

10 min before reactivation

100 102 86 60 180 85

87 96 111 41 5.5 32

ND ND ND 10 41 15

VOL. 178, 1996

FAMILY OF EXTRADIOL CATECHOL DIOXYGENASES

5255

FIG. 6. Hypothetical model for domain structures of three classes of extradiol catechol dioxygenases. The iron(II) ligands identified in the Pseudomonas strain LB400 dioxygenase BphC by X-ray crystallography (His-146, His-210, and Glu-260) are underlined (10). The positions marked by asterisks correspond to seven amino acid residues identified in the Pseudomonas strain LB400 enzyme as being situated close to the iron(II) cofactor, namely, the three iron(II) ligands and His-195, His-241, Ser-248, and Tyr-250 (10). Residues which are identically conserved in each class of amino acid sequences are in boldface.

gene has revealed that the inferred amino acid sequence of MhpB has 58% sequence identity with MpcI. Expression and purification of the MpcI protein have revealed that it shares a number of biochemical properties with MhpB: weak binding of the iron(II) cofactor, reactivation of the apoenzyme by iron(II) and ascorbate, broad specificity towards 3-substituted catechols (optimum side chain, propionate), and inactivation by cysteine- and histidine-directed reagents. Hence, there is good evidence that these two enzymes are structurally and functionally related. Amino acid sequence alignments have also revealed weaker sequence similarity with P. paucimobilis protocatechuate 4,5dioxygenase (LigB), with E. coli 3,4-dihydroxyphenylacetate 2,3-dioxygenase (HpcB), and with human HAO. This new family of enzymes comprises all of the extradiol dioxygenases which had previously shown a lack of sequence similarity with the Pseudomonas catechol 2,3-dioxygenases. Four conserved histidine residues are found in this family of dioxygenases, suggesting that there are active-site histidine residues which might function as iron(II) ligands. This is supported by the observed inactivation of MhpB and MpcI by the histidinedirected reagent diethyl pyrocarbonate. Analysis of the MhpB and MpcI sequences suggests that these enzymes consist of two similar domains (Fig. 3), reminiscent of the two-domain structure of the Pseudomonas strain LB400 dioxygenase BphD identified by X-ray crystallography (10). The sequence of the single-domain dioxygenase from Rhodococcus strain P6 has previously been shown to align with the C-terminal domain of the Pseudomonas strain LB400 dioxygenase BphC (10); however, we have found that it also aligns with the N-terminal domain of the E. coli MhpB sequence (Fig. 4). On the basis of these sequence alignments, we suggest a model for the three classes of extradiol dioxygenases, which is shown in Fig. 6. The small Rhodococcus strain P6 enzyme typifies a single-domain enzyme with a molecular mass of 21 kDa (1, 13), designated class I. The Pseudomonas strain LB400 enzyme typifies a two-domain enzyme in which the C-terminal domain binds iron(II) and is catalytically active (10), designated class II. The E. coli MhpB enzyme typifies a two-domain enzyme in which the N-terminal domain contains conserved histidine residues, designated class III. There is less sequence conservation in the C termini of the class III enzymes, just as there is less sequence conservation in the N termini of the class II enzymes. The evolution of both class II and class III enzymes could be explained by the duplication of a class I gene followed by the mutation and loss of function of either the N-terminal

domain (to give class II) or the C-terminal domain (to give class III). On the basis of this model, the positions of the conserved residues in the N-terminal domain of the class III enzymes can be compared indirectly with the positions of active-site residues identified by X-ray crystallography in the C-terminal domain of the class II enzymes (Fig. 6). The conserved His-10 of MhpB corresponds in position to His-146 of the LB400 enzyme, which is one of the iron(II) ligands (10). His-53 and His-115 of MhpB correspond in position to His-195 and His241 of the LB400 enzyme, which are positioned close to the iron(II) cofactor (10). The other iron(II) ligands in the LB400 enzyme, His-210 and Glu-260, correspond to Asp-76 and Asp129 in the E. coli MhpB sequence, neither of which is a conserved residue. This suggests that there may be a slightly different arrangement of active-site residues in the class II and class III enzymes. ACKNOWLEDGMENTS This work was supported by a studentship (to E.L.S.) from the Engineering and Physical Sciences Research Council. We thank P. Fortnagel (University of Hamburg) for the gift of the MpcI expression construct JM109/pAE166 and I. Giles (University of Southampton) for assistance with amino acid sequence alignments. REFERENCES 1. Asturias, J. A., L. D. Eltis, M. Prucha, and K. N. Timmis. 1994. Analysis of three 2,3-dihydroxybiphenyl 1,2-dioxygenases found in Rhodococcus globerulus P6. J. Biol. Chem. 269:7807–7815. 2. Blakley, E. R., and J. F. Simpson. 1963. The microbial metabolism of cinnamic acid. Can. J. Microbiol. 10:175–185. 3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254. 4. Bugg, T. D. H. 1993. Overproduction, purification and properties of 2,3dihydroxyphenylpropionate 1,2-dioxygenase from Escherichia coli. Biochim. Biophys. Acta 1202:258–264. 5. Burlingame, R. 1983. Ph.D. thesis. University of Minnesota, Minneapolis. 6. Burlingame, R., and P. J. Chapman. 1983. Catabolism of phenylpropionic acid and its 3-hydroxy derivative by Escherichia coli. J. Bacteriol. 155:113– 121. 7. Candidus, S., K. H. van Pee, and F. Lingens. 1994. The catechol 2,3-dioxygenase gene of Rhodococcus rhodochrous CTM: nucleotide sequence, comparison with isofunctional dioxygenases and evidence for an active-site histidine. Microbiology 140:321–330. 8. Christiansen, W. G. 1926. Some derivatives of gallic acid and pyrogallol. J. Am. Chem. Soc. 48:1358–1365. 9. Doolittle, R. F. 1987. Of URFs and ORFs: a primer on how to analyze derived amino acid sequences, p. 10–17. University Science Books, Mill Valley, Calif. 10. Han, S., L. D. Eltis, K. N. Timmis, S. W. Muchmore, and J. T. Bolin. 1995. Crystal structure of the biphenyl-cleaving extradiol dioxygenase from a PCB-

5256

SPENCE ET AL.

degrading Pseudomonad. Science 270:976–980. 11. Harayama, S., M. Kok, and E. L. Neidle. 1992. Functional and evolutionary relationships among diverse oxygenases. Annu. Rev. Microbiol. 46:565–601. 12. Harayama, S., and M. Rekik. 1989. Bacterial aromatic ring-cleavage enzymes are classified into two different gene families. J. Biol. Chem. 264: 15328–15333. 13. Heiss, G., A. Stolz, A. E. Kuhm, C. Mu ¨ller, J. Klein, J. Altenbuchner, and H. J. Knackmuss. 1995. Characterization of a 2,3-dihydroxybiphenyl dioxygenase from the naphthalene sulfonate-degrading bacterium strain BN6. J. Bacteriol. 177:5865–5871. 14. Kabisch, M., and P. Fortnagel. 1990. Nucleotide sequence of metapyrocatechase I (catechol 2,3-oxygenase I) gene mpcI from Alcaligenes eutrophus JMP222. Nucleic Acids Res. 18:3405–3406. 15. Kohara, Y., K. Akiyama, and K. Isono. 1987. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell 50:495–508. 16. Malherbe, P., C. Ko¨hler, M. Da Prada, G. Lang, V. Kiefer, R. Schwarcz, H. W. Lahm, and A. M. Cesura. 1994. Molecular cloning and functional expression of human 3-hydroxyanthranilic acid dioxygenase. J. Biol. Chem. 269:13792–13797. 17. Noda, Y., S. Nishikawa, K. I. Shiozuka, H. Kadokura, H. Nakajima, K. Yoda,

J. BACTERIOL.

18. 19. 20. 21. 22. 23. 24.

Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki. 1990. Molecular cloning of the protocatechuate 4,5-dioxygenase genes of Pseudomonas paucimobilis. J. Bacteriol. 172:2704–2709. Pascal, R. A., Jr., and D. S. Huang. 1986. Reactions of 3-ethyl-catechol and 3-(methylthio)-catechol with catechol dioxygenases. Arch. Biochem. Biophys. 248:130–137. Roper, D. I., and R. A. Cooper. 1990. Subcloning and nucleotide sequence of the 3,4-dihydroxyphenylacetate (homoprotocatechuate) 2,3-dioxygenase gene from Escherichia coli C. FEBS Lett. 275:53–57. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467. Segel, I. H. 1993. Enzyme kinetics, p. 371–385. Wiley-Interscience, New York. Spence, E. L., G. J. Langley, and T. D. H. Bugg. Cis-trans isomerisation of a cyclopropyl radical trap catalysed by extradiol catechol dioxygenases: evidence for a semiquinone intermediate. J. Am. Chem. Soc., in press. Vogel, A. I. 1989. Vogel’s textbook of practical organic chemistry, 5th ed., p. 695–701. Longman, Harlow, United Kingdom.