Characterization of a gene cluster from Ralstonia eutropha JMP134 encoding metabolism of 4-methylmuconolactone

Gene 206 (1998) 53–62

Characterization of a gene cluster from Ralstonia eutropha JMP134 encoding metabolism of 4-methylmuconolactone Rainer W. Erb *, Kenneth N. Timmis, Dietmar H. Pieper Department of Microbiology, GBF-National Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany Received 18 July 1997; received in revised form 3 October 1997; accepted 3 October 1997; Received by A. Bernardi

Abstract A 2,585 bp chromosomal DNA segment of Ralstonia eutropha JMP134 (formerly: Alcaligenes eutrophus JMP134) which contains a gene cluster encoding part of the modified ortho-cleavage pathway encodes a putative transport protein for 4-methylmuconolactone, a novel 4-methylmuconolactone methylisomerase and methylmuconolactone isomerase. The putative 4-methylmuconolactone transporter, a protein with a calculated molecular mass of 45.8 kDa, exhibits sequence homology to other members of the major superfamily of transmembrane facilitators and shows the common structural motif of 12 transmembranespanning a-helical segments and the hallmark amino acid motif characteristic of the superfamily. Consistent with the novelty of the reaction catalyzed by 4-methylmuconolactone methylisomerase, no primary sequence homologies were found between this enzyme or its gene and other proteins or genes in the data banks, suggesting that this enzyme represents a new type of isomerase. The molecular mass of the native 4-methylmuconolactone methylisomerase was determined by gel filtration analysis to be 25±2 kDa. From the polynucleotide sequence of the gene, a molecular mass of 12.9 kDa was calculated and hence we predict a homodimeric quaternary structure. The high sensitivity of 4-methylmuconolactone methylisomerase to heavy metals and thiolmodifying reagents implicates the involvement of sulfhydryl groups in the catalytic reaction. The methylmuconolactone isomerase – calculated molecular mass 10.3 kDa – has a primary structure related to the classical muconolactone isomerases (EC 5.3.3.4) of Acinetobacter calcoaceticus, of two Pseudomonas putida strains and of Ralstonia eutropha JMP134, suggesting that these are all isoenzymes. Consistent with this proposal is the finding that the purified protein exhibits muconolactone-isomerizing activity. © 1997 Elsevier Science B.V. Keywords: Biodegradation; Modified ortho-cleavage pathway; 4-Methylmuconolactone methylisomerase; Methylmuconolactone isomerase

1. Introduction Three distinct ortho-cleavage routes are responsible for the metabolism of catechols in Ralstonia eutropha * Corresponding author. Tel.: +49 531 6181406; Fax: +49 531 6181411; e-mail: [email protected] Abbreviations: Ap, ampicillin; bla, gene encoding b-lactamase; bp, base pair(s); Cm, chloramphenicol; IPTG, isopropyl b--thiogalactopyranoside; Km, kanamycin; kb, kilobase(s) or 1000 bp; kDa, kilodalton(s); MCS, multiple cloning site; ML, methylmuconolactone; mmlH, gene encoding putative transport protein for 4-methylmuconolactone; mmlI, gene encoding 4-methylmuconolactone methylisomerase; mmlJ, gene encoding methylmuconolactone isomerase; MmlH, putative transport protein for 4-methylmuconolactone; MMLI, methylmuconolactone isomerase; MLMI, 4-methylmuconolactone methylisomerase; ORF, open reading frame; Tc, tetracycline; p, plasmid; X-Gal, 5-bromo-4-chloro-3-indolyl b--galactopyranoside; ∞, denotes a truncated gene at the indicated side. 0378-1119/97/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 5 65 - 9

strain JMP134 (formerly: Alcaligenes eutrophus JMP134). The first of these affects the metabolism of chlorocatechols, intermediates in the degradation of chlorophenoxyacetates and chlorobenzoates (Pieper et al., 1988; Vollmer et al., 1993). The second is responsible for the catabolism of catechol ( Kuhm et al., 1990; ¨ Schlomann et al., 1990) by enzymes of the classical 3-oxoadipate pathway. The third ortho-cleavage pathway is involved in the degradation of 4-methylcatechol (Pieper et al., 1985) and involves a 4-methylmuconolactone methylisomerase (Pieper et al., 1990) as a key enzyme. The latter route is especially noteworthy since methylsubstituted catechols are generally degraded via metacleavage, whereas ortho-cleavage leads to methyl-substituted 4-carboxymethylbut-2-en-4-olides (methylmuconolactones) as dead-end metabolites (Catelani et al., 1971; Knackmuss et al., 1976). The degradation of

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4-carboxymethyl-4-methylbut-2-en-4-olide (trivially 4methylmuconolactone, abbrev. 4ML) is of special interest because muconolactones carrying an alkyl substituent at C-4 cannot be degraded in a fashion analogous to that of the classical 3-oxoadipate pathway (Pieper et al., 1985). A 4-methylmuconolactone methylisomerase capable of converting 4ML to 3-methylmuconolactone (3ML) was initially characterized from Ralstonia eutropha strain JMP134 (Pieper et al., 1985, 1990) and has also been purified from nocardioform actinomycetes (Bruce et al., 1989). The function of this enzyme may be considered to compensate for the initial ‘incorrect’ cycloisomerization of 3-methylmuconate. In

both Ralstonia eutropha and the actinomycetes, 3ML is further degraded via 4-methyl-3-oxoadipate (Bruce and Cain, 1988; Pieper et al., 1985), and hence probably by reactions analogous to those of the classical 3-oxoadipate pathway. It has, however, been reasoned that isoenzymes of the 3-oxoadipate pathway catalyze this reaction sequence (Pieper et al., 1985). In contrast to bacterial degradation of 4-methylcatechol, 3-methylcis,cis-muconate is directly cycloisomerized to 3ML in fungi (Powlowski and Dagley, 1985) (Fig. 1). The 4-methylmuconolactone methylisomerase has been recruited into Pseudomonas sp. B13 FR1 (abbreviated FR1) – a 4-chlorobenzoate degrading derivative of

Fig. 1. Ortho-cleavage pathways for catechol and 4-methylcatechol. Enzymes involved are as follows: C12O I (or II ), catechol 1,2-dioxygenase type I (or II ); MCI, muconate cycloisomerase; CMCI, chloromuconate cycloisomerase; MLMI, 4-methylmuconolactone methylisomerase; MLI, muconolactone isomerase; MMLI, methylmuconolactone isomerase; ELH, 3-oxoadipate enol-lactone hydrolase; MELH, isoenzyme of 3-oxoadipate enollactone hydrolase modified for the metabolism of methyl-substituted analogs.

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the 3-chlorobenzoate degrading Pseudomonas sp. B13 – by transfer of a hybrid cosmid pFRC20P, a derivative of pLAFR3 that contains a 26-kb DNA fragment from the JMP134 chromosome. Whereas the parental strain FR1 accumulates 4ML from 4-methylbenzoate, the constructed strain FR1(pFRC20P) was able to mineralize 4-methylbenzoate and 4-methylphenol via the orthocleavage pathway (Rojo et al., 1987). However, as the recipient strain was already able to catabolize 3ML, it was not clear if only the gene encoding the 4-methylmuconolactone methylisomerase or also additional genes of this modified ortho-cleavage pathway essential for mineralization of the 4-substituted aromatic substrates were transferred. Deletion and subcloning analysis of the inserted R. eutropha DNA localized the region that encoded the MLMI to a segment 3 kb in length (Rojo et al., 1987). As a first step towards identifying the essential components of the modified ortho-cleavage pathway in R. eutropha JMP134 for 4-methylcatechol, we have characterized the 3 kb gene cluster that has been transferred to FR1 containing the gene of the key enzyme 4-methylmuconolactone methylisomerase. This study has revealed genes for a putative transport protein for 4ML (MmlH ), 4-methylmuconolactone methylisomerase (MLMI ) and methylmuconolactone isomerase (MMLI ), and has confirmed the novelty of the 4-methylmuconolactone methylisomerase.

2. Materials and methods 2.1. Bacterial strains, plasmids, and culture conditions The strains and plasmids used in this study are listed in Table 1. Escherichia coli strains were routinely cultured in Luria–Bertani (LB) medium (Sambrook et al., 1989) at 30 or at 37°C. Where appropriate, antibiotics (Sigma, Deisenhofen, Germany) were used for selection at the following concentrations: ampicillin, 100 mg/ml; chloramphenicol, 20 mg/ml; kanamycin, 100 mg/ml. In some cloning experiments LB agar was supplemented with 40 mg/ml 5-bromo-4-chloro-3-indolyl b--galactopyranoside ( X-Gal; Boehringer, Mannheim, Germany) and 0.4 mM isopropyl b--thiogalactopyranoside ( IPTG; Boehringer) for screening of transformants. 2.2. Recombinant DNA techniques Plasmid DNA isolation, restriction enzyme digestion, ligation and other recombinant DNA techniques were carried out according to published protocols (Sambrook et al., 1989). Transformation of E. coli strains was performed either by an optimized CaCl method ( Tang 2 et al., 1994) or by electroporation (Dower et al., 1988). In order to generate the series of deletion subclones pRE402,… pRE421, plasmid pRE401 was first digested with EcoRI and the recessed 3∞-ends thereby generated

Table 1 Bacterial strains and plasmids used in this study Strain or plasmid E.coli strains JM109 XL1-Blue BL21(DE3) Plasmids pLysS pFRC40P

pBluescript SK pRE401 pRE402 pRE403…pRE421 pRE422 pRE423

Description

Reference or source

recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi D(lac-proAB) recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F ∞ proAB lacI qZDM15 Tn10 ( Tcr)] hsdS gal (lcIts857 ind1 S am7 nin5 lac UV5-T7 gene1)

Yanish-Perron et al., 1985 Bullock et al., 1987

Cmr; derivative of pACYC184, contains T7 gene 3.5 for T7 lysozyme and promotor w 3.8 for T7 RNA polymerase Kmr Cmr Apr; derivative of pSUP2021, contains xylXYZLS genes of TOL plasmid pWW0 and a 2.7 kb SacI fragment with the mml gene cluster of Ralstonia eutropha JMP134 Apr; derivative of pUC19, a-lac/MCSa Apr; contains a 2.7 kb SacI fragment with the mml gene cluster inserted in the MCS of pBluescript SK Apr; identical to pRE401 but with NotI–EcoRI fragment of MCS deleted Apr; pRE402 deletion subclones with progressively more of the mml gene cluster deleted Apr; derivative of pRE402 with 202 bp Esp3I fragment of the mmlHIJ gene cluster (nucleotides 1244–1445) deleted Apr; derivative of pRE402 with 541 bp EagI fragment of the mmlHIJ gene cluster (nucleotides 270–810) deleted

Studier and Moffatt, 1986 Studier, 1991 Negoro et al. (submitted )

Stratagene This study This study This study This study This study

aMCS, multiple cloning site; Apr, ampicillin resistant; Cmr, chloramphenicol resistant; Kmr, kanamycin resistant; Tcr, tetracycline resistant.

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were filled in with thionucleotides to make them nuclease resistant. It was then cut with NotI to yield a 5∞-overhanging end adjacent to the MLMI-containing fragment and incubated with exonuclease III (exoIII ). Aliquots were removed at regular intervals and treated with S1 nuclease to remove the progressively longer single-stranded regions generated by exo III, as described by Henikoff (Henikoff, 1984). Resulting molecules were recircularized by treatment with T4 DNA ligase and transformed into E. coli XL1-Blue. 2.3. DNA sequencing and sequence analysis The nucleotide sequence was determined by the dideoxy chain-termination method (Sanger et al., 1977) using pRE402,… pRE421 as templates for Taq DNA polymerase-initiated cycle sequencing reactions with fluorescence-labeled dideoxynucleotide terminators (Applied Biosystems, Weiterstadt, Germany). The sequencing reactions were analyzed using an automated DNA sequencer (Model 373, Applied Biosystems). Sequencing was carried out in a Landgraf thermocycler (Landgraf, Langenhagen, Germany) according to the following cycle conditions: denaturation at 96°C for 15 s, and both primer annealing and primer extension at 60°C for 15 s and 4 min, respectively. The reaction mixtures were set up according to the manufacturer’s suggestions with the modifications that 10 pmol of primer were used and formamide was added to a final concentration of 2.5%. The sequence of the universal primer PT7Seq designed to sequence the first strand was 5∞-GTAATACGACTCACTATAGGGCG-3∞, corresponding to nucleotides 625–647 of the pBluescript SK sequence. Based on the sequence information obtained after having sequenced the first strand, the second strand was sequenced using 13 different primers targetting the first strand. The overlapping sequences obtained were assembled into one continuous sequence, and open reading frames (ORFs) within this sequence were determined with the aid of the Gene Works version 2.45 software (IntelliGenetics, Mountain View, CA, USA). Sequence analysis and alignments were done with the PC/Gene software package, version 6.85 (IntelliGenetics). Homology searches were carried out in in all major databases including the EMBL, GenBank and SWISS-PROT data bases using the NCBI BLAST E-mail server. 2.4. Resting cell assay E. coli BL21(DE3)[pLysS] derivatives harboring selected deletion subclones of pRE402 were cultured at 30°C in LB medium supplemented with chloramphenicol and ampicillin to an optical density at 600 nm of 0.3. The cultures were then induced for T7 RNA polymerase expression by further incubation in the presence of

0.4 mM IPTG for 60 min at 30°C. The cells were harvested, washed with 50 mM sodium phosphate buffer (pH 7.0), and resuspended in 1/20 volume of the same buffer. Cells were incubated with 0.5 mM 4ML at 30°C on a gyratory shaker and sampled at 15 min intervals. 2.5. Preparation of crude cell extracts E. coli BL21(DE3)[pLysS ] harboring pRE402 or its derivatives was cultured and induced for T7 RNA polymerase expression as described above. Cells were harvested, washed and resuspended in 1/50 volume of 50 mM Tris–HCl buffer (pH 7.5) prior to disruption by a single passage through a French press (Model SLMAminco #FA-078, SLM Instruments, Urbana, IL, USA) operated at a pressure of 138 MPa. The cell debris was removed by centrifugation at 50 000×g for 30 min at 4°C. The clear supernatant fluid was carefully decanted and used as crude extract. 2.6. Monitoring of 4-methylmuconolactone methylisomerase activity Isomerization of 4ML into 3ML by either resting cells or crude cell extracts (10 ml ) was monitored by highperformance liquid chromotography (HPLC ) using a Merck liquid chromatograph (Merck, Darmstadt, Germany) equipped with a UV-detector with a SC125/Licrospher 100 RP8 5 mm column (Bischoff, Leonberg, Germany) and an aqueous solvent system containing 0.1% of 85% ortho-phosphoric acid and 18% methanol. The disappearance of 4ML (initial concentration 0.5 mM ) and formation of 3ML were monitored at 210 nm. 2.7. Methylmuconolactone isomerase assay Assays of methylmuconolactone isomerase activity in crude cell extracts were performed at 25°C in 50 mM Tris–HCl buffer (pH 7.5), with 0.2 mM (4S, 5S/4R, 5R)-5-chloro-3-methylmuconolactone as substrate (Prucha et al., 1996). The formation of the reaction product trans-3-methyldienelactone was followed by monitoring the increase in adsorbance at 270 nm using a Shimadzu UV-2100 spectrophotometer equipped with a thermojacketed cuvette holder. 2.8. Biochemicals 4-Methylmuconolactone was prepared as described by Knackmuss et al. (1976) and (4S, 5S/4R, 5R)5-chloro-3-methylmuconolactone was prepared according to Pieper et al. (1993).

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2.9. Nucleotide sequence accession number The nucleotide sequence reported here will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X99639.

3. Results 3.1. Gene designation The analyzed DNA fragment containes a gene cluster encoding part of the modified ortho-cleavage pathway of R. eutropha strain JMP134 involved in methylmuconolactone degradation. The responsible genes were therefore designated mml. 3.2. Analysis of the nucleotide sequence The fragment known to encode MLMI was excised as a 2.7-kb SacI fragment from pFRC40P and cloned into pBluescript SK to yield pRE401. A series of deletion subclones pRE402… pRE421 was generated and both strands of the 2.7-kb SacI fragment containing 2585 bp of chromosomal DNA from R. eutropha JMP134 encoding the mml gene cluster were sequenced ( Fig. 2). Through sequence comparisons and expression experiments we could identify three genes, designated mmlH, mmlI and mmlJ. Gene mmlH has a length of 1287 nucleotides with an initiation codon ATG at nucleotides 50–52, preceded 6 bp upstream by a putative ribosome binding site (Shine and Dalgarno, 1974), 5∞-AGGAGA-3∞. The polypeptide sequence deduced from this gene consists of 428 residues with a calculated molecular mass of 45.8 kDa. The mmlI gene is 342 bp long and starts with an initiation codon ATG at nucleotides 1402–1404, preceded by a Shine/Dalgarno-like sequence, 5∞-AGAGGAG-3∞, 6 bp upstream of the start codon. The encoded protein consisting of 113 amino acids has a predicted molecular mass of 12 874 Da. Gene mmlJ is located further downstream with an ATG codon at positions 1783–1785, a ribosome-binding like sequence, 5∞-AAGGAGA-3∞, 5 bp upstream of the initiation codon, and has a length of 276 nucleotides. The deduced polypeptide consists of 91 residues with a calculated mass of 10 310 Da. 3.3. Expression of mmlI and mmlJ in E. coli The deletion subclones pRE402,… pRE421 were also used in expression experiments. Additionally, deletions whose exact positions were confirmed by sequencing were introduced into the genes mmlH and mmlI (pRE422 and pRE423, respectively, see Table 1) in order to experimentally identify and verify their functions. The various subclones of pRE401 with deletions extending

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into the distinct mml genes were introduced into E. coli host strain BL21(DE3)[pLysS], which permits induction of transcription from phage T7 late promotors (Studier, 1991). After induction, the activities of MLMI and MMLI were assayed (Fig. 3). Either resting cells or cell extracts were incubated with 4ML and analyzed directly by HPLC and UV spectroscopy for MLMI activity. Constructs with deletions extending into mmlI did not exhibit MLMI activity, indicating that the mmlI gene encodes MLMI ( Fig. 3). MMLI activity was assayed in crude cell extracts; deletion of the mmlJ gene resulted in complete loss of activity, and thus we inferred that mmlJ encodes MMLI.

3.4. Sequence comparisons and characteristics The deduced amino acid sequence of mmlH shows homology to a large group of transport proteins termed the major facilitator superfamily (Marger and Saier, 1993), including the recently identified PcaK protein of Pseudomonas putida (Harwood et al., 1994) (22.2% amino acid identity, 17.1% similarity; GenBank accession number U10895), and the corresponding PcaK protein of Acinetobacter calcoaceticus ( Kowalchuk et al., 1994) (17.1% amino acid identity, 16.9% similarity; GenBank accession number L05770). This superfamily possesses a common structural motif of 12 transmembrane-spanning a-helical segments. In addition, specific regions of conserved amino acids in the primary sequence of the transporters have been identified ( Henderson, 1990). The hydropathicity profile of MmlH ( Fig. 4) indicates that this protein is hydrophobic and may indeed span the membrane 12 times. Consistent with this finding is the prediction of 12 membraneassociated a-helices using the algorithm of Eisenberg et al. (1984). Both hydropathicity plot and predicted ahelices also reflect the typical topology found in the superfamily, i.e. the organization of the 12 putative transmembrane helices in bundles of six, separated by a hydrophilic linker. Moreover, MmlH possesses the hallmark amino acid motif characteristic of the superfamily in the predicted positions. Amino acids 74–79 (DRYGRA) match the consensus (D/N )RXGR(R/K ) ( Henderson, 1990) that has subsequently been generalized to (R/K )X (R/K ) (Marger and Saier, 1993) 2 or 3 predicted to occur in the cytoplasmic loop between membrane-spanning helices 2 and 3. Similarly, amino acids 282–286 (RIGRR) perfectly match the consensus RXGRR (Henderson, 1990) occurring between transmembrane-spanning peptides 8 and 9. Two additional versions of the less stringent sequence (R/K )X 2 or 3 ( R/K ) were found in MmlH at residues 134–138 ( RHRGK ) and 217–221 (RKYAR), respectively. Finally, the hydrophilic region directly following the last membrane-spanning helix at positions 399–402

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Fig. 2. The mml gene cluster. The nucleotide sequence and deduced amino acid sequences of the chromosomal 2585 bp DNA fragment from Ralstonia eutropha JMP134 (formerly: Alcaligenes eutrophus JMP134) containing the genes mmlH, mmlI and mmlJ encoding a putative transport protein (MmlH ) for 4-methylmuconolactone, a novel 4-methylmuconolactone methylisomerase (MLMI ) and methylmuconolactone isomerase (MMLI ), respectively, are shown. Putative ribosome binding sites are underlined. The sequence has been deposited in the EMBL/GenBank data library and assigned accession number X99639.

( ETRG) fits well with the conserved sequence ETKG predicted at this position (Henderson, 1990). In contrast to MmlH, no gene or protein sequences

exhibiting significant sequence homology to the mmlI gene or its deduced protein were found in a comprehensive data base search. This indicates that the MLMI is

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Fig. 3. Deletion analyses of the mml gene cluster. Maps of the plasmid pRE402 and its deletion subclones are shown, as are the enzymatic activities exhibited by the deletion subclones. The direction of transcription is indicated by arrowheads. MLMI, 4-methylmuconolactone methylisomerase; MMLI, methylmuconolactone isomerase; nt, not tested.

Fig. 4. Hydropathicity plot of the putative 4-methylmuconolactone transporter MmlH. The algorithm of Kyte and Doolittle (1982) was used, with an interval of 9 amino acids.

not an isoenzyme of other isomerases, but rather a new type of isomerase, which is also consistent with the novelty of the MLMI-catalyzed reaction.

The deduced polypeptide sequence of mmlJ shares 98% (44 of 45 amino acids) identity with the aminoterminal amino acid sequence of the MMLI (Prucha et al., 1997) and the calculated molecular mass of 10.3 kDa is consistent with the 10±0.5 kDa value determined for the purified protein (Prucha et al., 1997). An alignment with three muconolactone isomerases ( EC 5.3.3.4), the classical isomerases acting on the nonmethylated muconolactones, of Acinetobacter calcoaceticus and two Pseudomonas putida strains shows amino acid identity in 38.1% and similarity in 37.1% of all positions (Fig. 5). Similarly, a binaric alignment of the MMLI with the 48 amino-terminal amino acids of the recently identified muconolactone isomerase of R. eutropha JMP134 (Prucha et al., 1996) (SWISS-PROT accession number of the partial amino acid sequence: PR0573) revealed amino acid identity in 45%, and similarity in 16.7% of all positions. These sequence comparisons indicate that the MMLI of R. eutropha JMP134 has a primary structure related to the classical muconolactone isomerases, suggesting that these are all

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Fig. 5. Multiple amino acid sequence alignment of methylmuconolactone isomerase and other muconolactone isomerases (EC 5.3.3.4). Sequences and the respective GenBank/EMBL data library accession numbers. 1, A. calcoaceticus, M76991 (Neidle et al., 1989); 2, P. putida RB1, M19460 (Aldrich and Chakrabarty, 1988); 3, P. putida PRS2000, U12557 (Houghton et al., 1995); 4, MMLI of R. eutropha JMP134 (formerly: A. eutrophus JMP134), X99639 (this study). The sequences were aligned using the multiple alignment program CLUSTAL (Higgins and Sharp, 1988). Amino acids identical in all proteins are indicated by asterisks, well conserved residues by periods.

isoenzymes. Consistent with this proposal is the finding that the purified protein exhibits muconolactone-isomerizing activity (Prucha et al., 1997). 3.5. Biochemical characteristics of MLMI Pieper et al. (1990) have reported previously that chelating agents, such as EDTA, had no effect on the MLMI activity, whereas CuSO significantly inhibited 4 the enzyme. In addition, p-chloromercuribenzoate, which modifies cysteine side chains in enzymes, also showed an inhibitory effect on the isomerase that could be reversed by addition of excess dithiothreitol. Taken together, these data suggest the involvement of sulfhydryl groups in the catalytic reaction. Indeed, there are two cysteine residues in the deduced amino acid sequence of the MLMI at positions 23 and 67, respectively (Fig. 2). The molecular mass of the native MLMI has reproducibly been determined by gel filtration analysis to be 25±2 kDa. Surprisingly, in SDS/PAGE a protein band of 40 kDa was dominant whenever a high isomerase activity was found (Pieper et al., 1990). Since a molecular mass of 12.9 kDa was calculated from the sequence, we predict a homodimeric quaternary structure for MLMI. Given the tetrameric structure of the Rhodococcus isomerase (Bruce et al., 1989), however, it cannot be ruled out that the MLMI of strain JMP134 might also be a homotetrameric protein, composed of two dimeric units, each of which is catalytically competent.

4. Discussion The DNA fragment studied here encodes part of the modified ortho-cleavage pathway of Ralstonia eutropha JMP134 that is responsible for the metabolism of methylaromatics (Pieper et al., 1985). The 2585 bp fragment contains three genes encoding a putative trans-

port protein (MmlH ) for 4-methylmuconolactone (4ML), the novel 4-methylmuconolactone methylisomerase (MLMI ) and methylmuconolactone isomerase (MMLI ), respectively. This gene cluster seems to form an operon that, as we inferred from the absence of a cycloisomerase and the presence of a transporter for 4ML, is responsible for the metabolism of 4ML. The putative operon includes MLMI as a key enzyme, together with at least one isoenzyme of the 3-oxoadipate pathway, the MMLI. Investigations to determine whether or not an isoenzyme of 3-oxoadipate enollactone hydrolase is also involved are currently in progress. Several lines of evidence indicate that the MmlH protein from Ralstonia eutropha JMP134 is a transporter for 4ML. Although the protein does not exhibit a very high degree of sequence homology to other transport proteins, it shows the common structural motif characteristic of the major superfamily of transmembrane facilitators (Marger and Saier, 1993). In addition, it is located directly upstream of the mmlI gene, the first gene of the putative new mml operon, whose substrate is 4ML. Lactones are common in nature (Rousseau, 1995) but passive diffusion across biological membranes may limit their rate of metabolism by saprophytic microorganisms. The existence of specific transporters for several aromatic acids, including 4-chlorobenzoate, 4-hydroxybenzoate, mandelate, and the xenobiotic 4-toluene sulfonate which has been postulated in several studies (Allende et al., 1992; Groenewegen et al., 1990; Harwood et al., 1994; Higgins and Mandelstam, 1972; Locher et al., 1993) is indicative that the MmlH protein described here could well be a lactone transporter of Ralstonia eutropha JMP134. Methylmuconolactone methylisomerases have until now only been found in the nocardioform actinomycete Rhodococcus rhodocrous N75 (Bruce et al., 1989) and the Gram-negative bacterium Ralstonia eutropha JMP134 (Pieper et al., 1990). A lack of nucleotide sequence homology of mmlI of R. eutropha JMP134 and

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of amino acid sequence homology of MmlH to known genes or proteins is consistent with the novelty of the MLMI-catalyzed reaction. Pieper et al. (1990) postulated a reaction mechanism for MLMI involving a covalently bound transition state intermediate resulting from an attack of the lactone ring of 4ML at C-4 by an enzyme nucleophile. Subsequent ring closure in the alternate direction generates a new lactone structure in which the methyl group, although linked to the same carbon atom, has formally changed its relative position. The high sensitivity of the isomerase to heavy metals and thiol-modifying reagents implies that the enzyme nucleophile might be a sulfhydryl group of one of the cysteines in the active site of the enzyme. Current experiments to substitute these cysteines by site-directed mutagenesis should provide additional information on this point. In contrast to the MLMI, which represents a novel type of isomerase, the MMLI is an isoenzyme of the muconolactone isomerases of the classical 3-oxoadipate pathway. As described recently by Prucha et al. (1997), the biochemical properties of these enzymes are quite similar and the MLMI also exhibits muconolactoneisomerizing activity. This isomeric muconolactone isomerase is thus of special interest in terms of evolutionary comparisons with the recently described muconolactone isomerase of R. eutropha JMP134 (Prucha et al., 1996) and the extensively characterized muconolactone isomerases of Pseudomonas putida (Aldrich and Chakrabarty, 1988; Houghton et al., 1995) and Acinetobacter calcoaceticus (Neidle et al., 1989). This characterization of the mml gene cluster suggests that the modified ortho-cleavage pathway of Ralstonia eutropha JMP134 includes at least two different operons, one encoding the classical type I enzymes of the 3-oxoadipate pathway that transform 4-methylcatechol to 4ML, and a second one, described here, encoding enzymes which further degrade 4ML to 4-methyl3-oxoadipate or 4-methyl-3-oxoadipate-enol-lactone. The MLMI represents a key function in the sense that it transforms 4ML into the isomeric 3ML, thereby solving the metabolic problem of how to transform 4ML to (methyl ) 3-oxoadipate.

Acknowledgement We thank Bettina Hofer for helpful discussions, and ¨ ¨ A. Arnscheidt, A. Kruger and C. Strompl for technical assistance with the sequencing. This work was supported ¨ by the Bundesministerium fur Bildung, Wissenschaft, Forschung und Technologie (BMBF grant 0319433B). K.N.T. gratefully acknowledges support from the Fonds der Chemischen Industrie.

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