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∗ This work was supported by Grant SAF94-0892 from Comisión Interministerial de Ciencia y Tecnologa. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X81372. ‡ Recipient of a fellowship from Fundación para la Investigación Cientfica y Técnica (Asturias-Spain).
A full-length cDNA coding for a novel human serine hydrolase has been cloned from a breast carcinoma cDNA library. Nucleotide sequence analysis has shown that the isolated cDNA contains an open reading frame coding for a polypeptide of 274 amino acids and a complete Alu repetitive sequence within its 3′-untranslated region. The predicted amino acid sequence contains the Gly-X-Ser-X-Gly motif characteristic of serine hydrolases and displays extensive similarity to several prokaryotic hydrolases involved in the degradation of aromatic compounds. The highest degree of identities was detected with four serine hydrolases encoded by the bphD genes of different strains of Pseudomonas with the ability to degrade biphenyl derivatives. On the basis of these sequence similarities, this novel human enzyme has been tentatively called Biphenyl hydrolase-related protein (Bph-rp). The Bph-rp cDNA was expressed in Escherichia coli, and after purification, the recombinant protein was able to degrade p-nitrophenylbutyrate, a water-soluble substrate commonly used for assaying serine hydrolases. This hydrolytic activity was abolished by diisopropyl fluorophosphate, a covalent inhibitor of serine hydrolases, providing additional evidence that the isolated cDNA encodes a member of this protein superfamily. Northern blot analysis of poly(A)+ RNAs isolated from a variety of human tissues revealed that Bph-rp is mainly expressed in liver and kidney, which was also confirmed at the protein level by Western blot analysis with antibodies raised against purified recombinant Bph-rp. According to structural characteristics, hydrolytic activity and tissue distribution of Bph-rp, a potential role of this enzyme in detoxification processes is proposed.
The serine hydrolases are defined as a functional class of hydrolytic enzymes that contain a serine residue in their active site. This enzyme class is composed of a large number of diverse members, including serine proteinases, esterases, and lipases, which are able to hydrolyze a wide variety of substrates, thus performing vastly different biological roles. However, despite this marked variability, the proteins belonging to this superfamily of serine-dependent hydrolytic enzymes can be grouped into subfamilies with closely related members in terms of substrate specificity or amino acid sequence similarities (
). This is the case of a recently described group of serine hydrolases isolated from diverse bacterial strains, which display significant amino acid sequence similarities and whose common function would be the degradation of aromatic compounds (
Over the last few years, the employment of soil bacteria capable of utilizing a wide range of aromatic compounds as sources of carbon and energy has raised considerable interest in biodegradation processes, since many of these compounds are widespread and persistent environmental pollutants (
). These microorganisms employ a number of different enzymes for the initial attack on the structurally diverse aromatic substrates, but the catabolic pathways tend to converge on just a few central intermediates such as catechol or substituted catechols. These key intermediates are subsequently cleaved and processed by one of a few central pathways to Krebs cycle intermediates. For example, biphenyls and their polychlorinated derivatives, toluene, phenol, or xylenes, are all metabolized to catechol by different enzymatic reactions, but the conversion of catechol to Krebs cycle intermediates is mediated by common enzymatic steps called the metacleavage pathway (
). One of the key reactions of these aromatic catabolic routes is catalyzed by a serine hydrolase with the ability to perform the hydrolytic cleavage of carbon-carbon bonds, one of the rarest of all enzyme reaction types (
). At present, seven bacterial hydrolases catalyzing this type of reaction have been characterized at the amino acid sequence level. Four of them, named 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolases, are the products of the respective bphD genes from four different strains of Pseudomonas: Pseudomonas sp strain KKS102 (
), and Pseudomonas testosteroni B-356 (GenBankTM accession number L34338). These hydrolases are involved in the degradation of biphenyls and their polychlorinated derivatives and show a high degree of amino acid sequence similarity. These four enzymes are also significantly related to dmpD, todF, and xylF, the equivalent hydrolases of the metacleavage pathways responsible for the degradation of phenol and methyl-substituted phenols (
), by different bacterial strains. All seven proteins share about 25% identical residues, including the Gly-X-Ser-X-Gly sequence encompassing the active site Ser of this enzyme class, which suggests that despite their discrete substrate specificities, all of them derive from a common ancestor, which was subsequently adapted to degrade the different aromatic compounds.
During the course of studies directed to look for biochemical markers that could be of interest in breast cancer (
) we have cloned from a human breast cancer cDNA library a novel human serine hydrolase with a high degree of sequence similarity to the above mentioned prokaryotic proteins involved in the degradation of aromatic compounds. In this work, we present the molecular cloning and nucleotide sequence of this novel enzyme named biphenyl hydrolase-related protein (Bph-rp).1(
We also analyze its expression in human tissues and perform a functional analysis with the protein produced in Escherichia coli. Finally, on the basis of the obtained results, we propose a potential role of this enzyme in detoxification processes.
MATERIALS AND METHODS
A human breast carcinoma cDNA library constructed in λgt11 and two Northern blots containing RNAs from many different human tissues were purchased from Clontech (Palo Alto, CA). Oligonucleotides were synthesized by the phosphoramidite method in an Applied Biosystems DNA synthesizer, model 381 A, and used directly after synthesis. Restriction endonucleases and other reagents used for molecular cloning were from Boehringer Mannheim. Double-stranded DNA probes were radiolabeled with [32P] dCTP (3000 Ci/mmol) from Amersham Corp. using a commercial random priming kit purchased from Pharmacia Biotech Inc. p-Nitrophenylbutyrate and diisopropyl fluorophosphate were purchased from Sigma. Reagents for amino acid sequencing were from Applied Biosystems (Foster City, CA).
Screening of a Human Breast Carcinoma cDNA Library
About 1 × 106 pfu of a human breast carcinoma cDNA library were plated using E. coli Y1088 as host and analyzed according to standard procedures (
). The probe was generated by PCR amplification of DNA isolated from the human breast carcinoma cDNA library with primers 5′-TGGAGTGAGAGAACAAG-3′ and 5′-GCAAAACGCAAATGCAG-3′. Hybridization to the radiolabeled probe was carried out at 55°C for 18 h in 6 × SSC (1 × SSC = 150 mM NaCl, 15 mM sodium citrate, pH 7.0), 5 × Denhardt's solution (1 × Denhardt's solution = 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, 0.02% Ficoll), 0.1% SDS, and denatured herring sperm DNA (100 μg/ml). The membranes were washed twice for 1 h at 60°C in 1 × SSC, 0.1% SDS and exposed to XAR-5 film (Kodak) at −70°C with intensifying screens. Following plaque purification, the cloned insert was excised by EcoRI digestion, and the resulting fragments were subcloned into the EcoRI site of pEMBL19.
Selected DNA fragments were inserted in the polylinker region of phage vector M13 mp19 (
) using either M13 universal primer or cDNA-specific primers and the Sequenase Version 2.0 kit (U. S. Biochemical Corp.). All nucleotides were identified in both strands. Sequence ambiguities were solved by substituting dITP for dGTP in the sequencing reactions according to the instructions of the manufacturer of the kit. Computer analysis of DNA and protein sequences was performed with the software package of the University of Wisconsin Genetics Computer Group (
A 995-bp DNA fragment containing the complete coding sequence for Bph-rp was obtained by PCR amplification of the isolated full-length cDNA with primers 5′-ATGCCCAGGAATCTGCT-3′ and 5′-CTGAAGCTAGAGGCTGT-3′. The PCR reaction was carried out for 30 cycles of denaturation (95°C for 1 min), annealing (48°C for 1 min), and extension (72°C for 1 min). The PCR product was phosphorylated with T4 polynucleotide kinase and ligated to the expression vector pET3c (
), previously treated with NdeI and nuclease S1. The resulting plasmid, called pETX3, was transformed into E. coli strain BL21(DE3), and the transformed cells were grown in LB broth containing 100 μg ampicillin/ml at 37°C for about 16 h, diluted 1:100 with the same medium, and grown to an A600 of 1.0. Then isopropyl-1-thio-β-D-galactopyranoside was added to a final concentration of 1 mM, and the incubation was continued for another 3 h. Cells were collected by centrifugation, resuspended in volume of phosphate-buffered saline, lysed by using a French press, and centrifuged at 20,000 × g for 20 min at 4°C. The protein was purified by DEAE-chromatography in 50 mM Tris, pH 7.4, and the retained material was eluted with a gradient of 50 mM Tris, pH 7.4, 0.5 M NaCl. Fractions containing Bph-rp, as judged by SDS-polyacrylamide gel electrophoresis, were pooled and gel chromatographed on a Superdex 75 HR 10/30 column (Pharmacia Biotech Inc.) in 0.1 M ammonium acetate, pH 6.5, at a flow rate of 0.1 ml/min and using a SMART system chromatograph (Pharmacia Biotech Inc.). Fractions of 250 μl were collected, and those containing pure recombinant Bph-rp were pooled and lyophilized.
Enzyme Activity Measurements
The enzyme activity of purified Bph-rp was measured using 0.5 mMp-nitrophenylbutyrate as substrate and following the procedure described by
with minor modifications. Assays were performed at 37°C, in 100 mM sodium phosphate buffer, pH 7.0, 90 mM NaCl, and the activity was measured at 410 nm by a UV-visible recorder spectrophotometer UV-160 (Shimadzu). For inhibition assays, the reaction mixture was preincubated with 0.1 mM diisopropyl fluorophosphate at 37°C for 1 h, and the remaining activity was determined using p-nitrophenylbutyrate as above. Diisopropyl fluorophosphate interaction with the putative serine residue of the Bph-rp active center was examined by incubating 15 μg of recombinant protein with 2 μl of [1,3-3H]diisopropyl fluorophosphate (10 Ci/mmol) (Amersham), for 1 h at 37°C. Then, 20 μl of 50 mM unlabeled diisopropyl fluorophosphate was added, and the samples were electrophoresed in 13% SDS-polyacrylamide gel. The gel was then fixed with 20% trichloroacetic acid and washed for 30 min with water to remove the excess trichloroacetic acid. Finally, the gel was incubated in 1 M sodium salicylate, dried, and exposed to XAR-5 film (Kodak) at −70°C.
Amino Acid Sequence Analysis
Purified recombinant Bph-rp or tryptic peptides generated from the purified protein were subjected to automatic Edman degradation on an Applied Biosystems 477A sequencer in the presence of Polybrene. The anilinothiazolinones were converted to phenylthiohydantoin derivatives in the automatic conversion flask of the sequencer and identified and quantified with an on-line phenylthiohydantoin analyzer (model 120 A; Applied Biosystems). The tryptic peptides were obtained after treatment of purified Bph-rp with trypsin for 16 h at 37°C at an enzyme:substrate ratio of 1:50 (w/w). The resulting fragments were fractionated by reverse-phase HPLC on a μRPC C2/C18 column (Pharmacia Biotech Inc.) equilibrated with 0.1% trifluoroacetic acid. The runs were carried out in a SMART system fast protein liquid chromatography at room temperature and at a flow rate of 0.1 ml/min. Peptides were eluted with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid, and their absorbance was monitored at 214 nm.
Northern Blot Hybridization
Northern blots containing 2 μg of poly(A)+ RNA of different human tissue specimens were prehybridized at 42°C for 3 h in 50% formamide, 5 × SSPE (1 × SSPE = 150 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA, pH 7.4), 10 × Denhardt's solution, 2% SDS, and 100 μg/ml of denatured herring sperm DNA and then hybridized with radiolabeled Bph-rp cDNA (a 995-bp PCR fragment extending from bp 213 to 1207) for 20 h under the same conditions. Filters were washed with 0.1 × SSC, 0.1% SDS for 2 h at 50°C and exposed to autoradiography. RNA integrity and equal loading was assessed by hybridization with an actin probe as indicated by the manufacturer.
Antiserum Production and Western Blot Analysis
200 μg of purified Bph-rp was used to immunize a New Zealand White rabbit according to the method described by
. The rabbit was bled 6 weeks after the injection of the recombinant protein, and IgGs were purified by anion exchange chromatography on a DEAE-cellulose column (Whatman DE52), equilibrated and eluted with 20 mM phosphate buffer, pH 7.2. Finally, the obtained antibodies (diluted 1:1000) were used for Western blot analysis as described previously (
Isolation and Characterization of a cDNA Encoding Human Biphenyl Hydrolase-related Protein
During the course of studies directed to examine the possible relevance of human milk-fat globule membrane proteins as tumor markers in breast cancer, we noted that a partial cDNA sequence putatively encoding one of these proteins, with a molecular mass of 70 kDa and sharing several properties with bovine butyrophilin, was not related to the amino acid sequence of this protein (
) prompted us to examine the possibility that the reported nucleotide sequence could encode a different protein. To do that, we first screened a human breast cancer cDNA library with a probe generated by PCR using synthetic oligonucleotides derived from the partial cDNA sequence proposed to encode the 70-kDa human milk-fat globule protein (
). Upon screening of approximately 1 × 106 plaque-forming units, two positive clones named x-14 and x-15 were identified. Nucleotide sequence analysis of both clones revealed that the sequence corresponding to x-15 was entirely contained within that obtained for x-14, indicating that it was a partial cDNA clone. Further analysis of the sequence of the largest clone showed an open reading frame 822 bp long, starting with an ATG codon at position 213 and ending with a TGA codon at position 1037 (Fig. 1). This open reading frame codes for a protein of 274 amino acid residues with a calculated molecular weight of 31,106.
A comprehensive search of the EMBL and GenBankTM nucleic acid data bases revealed that this human sequence had significant homology with those from a group of bacterial proteins involved in the degradation of aromatic compounds, including biphenyl derivatives, phenol, toluene, and xylene (
). A more detailed search for sequence similarities between the identified amino acid sequence and those corresponding to these prokaryotic enzymes revealed that the highest degree of identities (about 25%) was detected with four different serine hydrolases (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolases) encoded by the bphD genes of different strains of Pseudomonas (Fig. 2). The percentage of identities of the human protein with the remaining members of this family of bacterial serine hydrolases was slightly lower, ranging from 20% with dmpD to 22% with xylF, the equivalent hydrolases (2-hydroxymuconic-semialdehyde hydrolases) of pathways responsible for the degradation of phenol and xylene, respectively. However, in all cases the sequence similarity extends throughout the complete amino acid sequence of these proteins, being of special relevance to the occurrence of some regions with a high degree of conservation in all of them (Fig. 2). Thus, the human sequence contains at an equivalent position the Gly-X-Ser-X-Gly consensus motif that is a characteristic feature of serine hydrolases from different sources (
). The serine residue contained in this consensus sequence has been proposed to be the essential catalytic residue in the above mentioned hydrolases involved in the degradation of aromatic compounds. Consequently, it is tempting to speculate that the Ser-122 of the identified open reading frame would correspond to the catalytic serine residue of this putative novel human serine hydrolase that, according to its sequence similarity to bacterial proteins encoded by bph genes, has been tentatively called human Biphenyl hydrolase-related protein (Bph-rp).
The amino acid sequence alignment of Bph-rp with these prokaryotic serine hydrolases could also be helpful to identify the histidyl and acidic residues that together with Ser-122 could form the catalytic triad found to occur in proteins belonging to the serine hydrolase superfamily. Thus, although at present there is not experimental evidence on the identity of catalytic residues in these prokaryotic hydrolases,
have proposed that two conserved aspartic acid and histidyl residues could be part of the catalytic triad in 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase from Pseudomonas sp KKS102 and in 2-hydroxymuconic-semialdehyde hydrolase from P. putida. Both residues are perfectly conserved in the human sequence (positions 227 and 255) as well as in the remaining members of the bacterial protein family related to Bph-rp (Fig. 2 and data not shown). Consequently, these residues would be the most likely candidates to form part of the putative catalytic triad presumed to be essential for the enzymatic activity of both human and microbial enzymes. The ongoing studies on the three-dimensional structure of Bph-rp will contribute to confirm this prediction and to elucidate whether this human enzyme belongs to the α/β hydrolase fold family of proteins as already proposed for its bacterial homologues (
Finally, and also in relation to the structural analysis of the nucleotide sequence of the cDNA coding for Bph-rp, the occurrence of an Alu repetitive sequence in the 3′-noncoding sequence of the isolated cDNA clone is noteworthy. This Alu element is oriented in the same transcriptional direction as the Bph-rp gene, beginning at nucleotide 1207 and terminating at the poly(A)-rich region of the 3′-end of the Bph-rp cDNA (Fig. 1). Computer analysis of diagnostic nucleotides at specific positions in Alu sequences (
). The occurrence of this ancient Alu sequence in the 3′-flanking region of the mRNA for human Bph-rp is very unusual, since despite the fact that the short interspersed elements constitute about 5% of the human genome, most of them have been found in intergenic DNA or in introns, being spliced out during processing to the mature RNAs (
). The biological significance of the occurrence of this Alu repeat in the 3′-nontranslated region of the human Bph-rp mRNA as well as in those equivalent cases including mRNAs for human low density lipoprotein receptor (
) remains to be determined. In this regard, it should be remembered that Alu sequences have a high degree of similarity with the elongation arrest domain of 7SL RNA, a component of the signal recognition particle involved in protein secretion (
). On the basis of this homology, the Alu J element here detected within the Bph-rp mRNA could be important in the translational modulation of Bph-rp expression, as already proposed for Alu transcripts that are transported to the cytoplasmic compartment (
Production of Recombinant Biphenyl Hydrolase-related Protein in E. coli
As a preliminary step to examine the functional role of Bph-rp as well as to obtain specific antibodies against the protein that could be useful for further studies of the native protein in human tissues, we undertook the production of this protein in E. coli. To do that, a 995-bp fragment containing the entire Bph-rp open reading frame was first subcloned in the T7-RNA polymerase-based expression vector pET3c developed by
. The resulting plasmid, named pETx3, was transformed into E. coli strain BL21(DE3), and the transformed bacteria were induced to produce the recombinant protein by treatment with isopropyl-1-thio-β-D-galactopyranoside. Extracts were prepared from the induced bacteria, and their protein composition was examined by SDS-PAGE. As shown in Fig. 3, the soluble fraction of the extract contained a major protein of about 30 kDa, which was absent in the extracts from bacteria carrying the control plasmid. This recombinant protein was isolated from the bacterial extracts using the strategy outlined under “Materials and Methods.” Thus, extracts containing about 50 mg of total protein were first chromatographed on a DEAE anion exchange column. As judged by SDS-PAGE, the recombinant Bph-rp was detected in the flow-through fraction of the column, indicating that the protein did not bind to this chromatographic support. The material containing the putative Bph-rp was then fractionated by size exclusion fast protein liquid chromatography and the eluate material analyzed by 280-nm absorption and SDS-PAGE (Fig. 3 and 4A). The main peak of the chromatogram corresponded to an isolated, single polypeptide chain protein, with the expected molecular mass of 30 kDa. In order to confirm the identity of this purified recombinant protein as human Bph-rp, 25 μg of isolated protein were subjected to automatic Edman degradation. However, no phenylthiohydantoins could be identified in two separate runs, suggesting that the purified protein was blocked at its N-terminal end. Then in order to obtain information regarding internal amino acid sequences, the recombinant protein was treated with trypsin, and the resulting peptides were separated by reverse-phase HPLC in a C2/C18 column (Fig. 4B). Automatic Edman degradation of a series of isolated tryptic peptides (T18, Tyr-Pro-Ser-Tyr-Ile-His-Lys; T19, Asp-Val-Ser-Lys; T25, Trp-Val-Asp-Gly-Ile-Arg) revealed that all of them had sequences that could be matched with regions present in the amino acid sequence predicted from the cDNA sequence determined for human Bph-rp (Fig. 1). According to these data, we can conclude that the 30-kDa polypeptide produced in E. coli corresponds to human Bph-rp.
In order to examine the enzymatic activity of this recombinant protein, 2 μg of isolated Bph-rp was incubated with p-nitrophenylbutyrate, which is a commonly used water-soluble substrate for members of the serine hydrolase protein superfamily. In addition, this aromatic ester may somewhat resemble the aromatic compounds metabolized by the prokaryotic enzymes structurally related to Bph-rp. The recombinant Bph-rp displayed an hydrolytic activity on p-nitrophenylbutyrate (1.2 nmol/min/μg protein) that was fully abolished by diisopropyl fluorophosphate, an inhibitor of serine hydrolases. Furthermore, incubation of purified Bph-rp with radioactive diisopropyl fluorophosphate followed by SDS-PAGE/fluorography analysis, demonstrated that this active-site reagent was able to bind to the recombinant protein (Fig. 3B). Taken together, these results provide a strong indication that Bph-rp is an authentic serine hydrolase. The degree of activity of Bph-rp on p-nitrophenylbutyrate is similar to that reported for bovine milk lipoprotein lipase against the same substrate (1.0 nmol/min/μg protein) (
). These data suggest that the natural substrates of Bph-rp are distantly related to p-nitrophenylbutyrate. In this regard, it should also be mentioned that studies directed to examine the possibility that some available biphenyl derivatives (kindly provided by Dr. V. de Lorenzo) could be more appropriate substrates for Bph-rp have failed to demonstrate significant levels of hydrolytic activity, suggesting that despite its structural similarity to polychlorinated biphenyl hydrolases from prokaryotic sources, this novel human enzyme may have specific requirements in terms of substrates or experimental conditions of analysis of its hydrolytic activity. Nevertheless, it is also possible that the human Bph-rp produced in bacteria may be only partially functional. Studies are in progress to examine these possibilities as well as to try to identify the natural substrates of this human enzyme and to precisely establish the kinetics of hydrolysis of these substrates.
Expression Analysis of the Gene Encoding Biphenyl Hydrolase-related Protein in Human Tissues
To examine the expression of the Bph-rp gene in human tissues, two Northern blots containing poly(A)+ RNAs obtained from different human tissues including leukocytes, colon, small intestine, ovary, testis, prostate, thymus, spleen, pancreas, kidney, skeletal muscle, liver, lung, placenta, brain, and heart, were hybridized with a fragment of the cDNA coding for Bph-rp, which lacked the 3′-flanking region containing the Alu repetitive sequence. As shown in Fig. 5, a strong hybridizing band of about 1.8 kilobases was detected in RNA from liver and kidney and at lower intensity in RNA from other tissues including intestine, heart, and skeletal muscle. It is also noteworthy that an additional hybridizing band of about 2.4 kilobases was recognized by the Bph-rp probe in some tissues, especially in the lane corresponding to RNA from kidney. Interestingly, this large band was absent in liver, the tissue in which the expression of the 1.8-kilobase Bph-rp transcript was maximal. The origin of these two transcripts is unclear, although it seems likely that they could be the result of alternative splicing events or alternative utilization of polyadenylation sites. However, the possibility of existence of additional genes with a high degree of sequence similarity with Bph-rp and differentially expressed in human tissues cannot be definitively ruled out. The expression analysis of Bph-rp in human tissues was also confirmed at the protein level by using antibodies raised in rabbits against the recombinant protein produced in E. coli. As shown in Fig. 3C, these antibodies recognize a protein in human liver extracts that has virtually the same molecular mass as recombinant Bph-rp. The close agreement between the molecular mass of Bph-rp deduced from SDS-PAGE mobility and that calculated from the amino acid sequence suggests that major posttranslational modifications like proteolytic processing or extensive glycosylation events are not occurring in Bph-rp. This last observation is also consistent with the absence of N-linked glycosylation consensus sequences in the amino acid sequence deduced in this work for human Bph-rp (Fig. 1). These data, together with the absence of any immunoreactive band of 70 kDa in the Western blot analysis of Bph-rp production by human tissues, strongly suggest that Bph-rp is not related to the 70-kDa mucin-associated antigen thought to be encoded by the partial cDNA used herein for cloning Bph-rp. Since this partial cDNA was isolated upon screening of a cDNA library with antibodies raised against the 70-kDa component of human milk-fat globule membranes (
), it seems likely that the previously isolated cDNA clone coding for the C-terminal end of Bph-rp was picked up as a result of an immunological cross-reactivity event between these two otherwise unrelated proteins.
The high level expression of human Bph-rp in liver and kidney, together with its structural relationship to bacterial enzymes involved in biodegradative pathways of different aromatic compounds, could be indicative of a potential role for this protein in detoxification processes. This role may be somewhat parallel to that proposed for other mammalian hydrolases, including nonspecific carboxylesterases (
). These proteins are also produced at high levels in liver and kidney, hydrolyze a variety of exogenous and endogenous substrates, and are thought to function mainly in drug metabolism and detoxification of many harmful substances (
have recently described that some of these mammalian hydrolases, especially epoxide hydrolases, display significant amino acid sequence similarity with the diverse bacterial proteins involved in the degradation of aromatic compounds that according to the present results are most closely related to human Bph-rp. The availability of the different reagents developed in the present work, including recombinant Bph-rp, and specific antibodies and probes for this novel human enzyme, will be useful for a better understanding of the functional significance of the structural similarities between these two sets of enzymes that diverged more than 1.5 billion years ago. Finally, these reagents will also be of interest to examine the significance of the production of Bph-rp by breast carcinomas and the possibility that it could play some role in the metabolism of aromatic endogenous substances like steroid hormones within the human mammary gland.
We thank Dr. S. Gascón for support; Drs. M. Balbn, A. M. Pendás, G. Velasco, L. M. Sánchez, and V. de Lorenzo for helpful comments; and Dr. S. G. Granda (Scientific Computer Center, Universidad de Oviedo) for computer facilities.