The Sequential Action of a Dipeptidase and a β-Lyase Is Required for the Release of the Human Body Odorant 3-Methyl-3-sulfanylhexan-1-ol from a Secreted Cys-Gly-(S) Conjugate by Corynebacteria
2008; Elsevier BV; Volume: 283; Issue: 30 Linguagem: Inglês
10.1074/jbc.m800730200
ISSN1083-351X
Autores Tópico(s)Neuropeptides and Animal Physiology
ResumoHuman axillary odor is formed by the action of Corynebacteria on odorless axilla secretions. Sulfanylalkanols, 3-methyl-3-sulfanylhexan-1-ol in particular, form one key class of the odoriferous compounds. A conjugate with the dipeptide Cys-Gly has been reported as the secreted precursor for 3-methyl-3-sulfanylhexan-1-ol. Here, we confirm the Cys-Gly-(S) conjugate as the major precursor of this odorant, with lower levels of the Cys-(S) conjugate being present in axilla secretions. The enzymatic release of 3-methyl-3-sulfanylhexan-1-ol from the Cys-Gly-(S) conjugate by the axilla isolate Corynebacterium Ax20 was thus investigated. Cellular extracts of Ax20 released 3-methyl-3-sulfanylhexan-1-ol from the Cys-Gly-(S) conjugate and from the Cys-(S) conjugate, whereas the previously isolated C-S lyase of this bacterial strain was only able to cleave the Cys-(S) conjugate. o-Phenanthroline blocked the release from the Cys-Gly-(S) conjugate but did not affect cleavage of the Cys-(S) conjugate, indicating that in a first step, a metal-dependent dipeptidase hydrolyzes the Cys-Gly bond. This enzyme was purified by four chromatographic steps and gel electrophoresis, and the partial amino acid sequence was determined. The corresponding gene was cloned and expressed in Escherichia coli. It codes for a novel dipeptidase with a high affinity toward the Cys-Gly-(S) conjugate of 3-methyl-3-sulfanylhexan-1-ol. Co-incubating either the synthetic Cys-Gly-(S) conjugate or fresh axilla secretions with both the C-S lyase and the novel dipeptidase did release 3-methyl-3-sulfanylhexan-1-ol, proving that the sequential action of these two enzymes from the skin bacterium Corynebacterium Ax20 does release the odorant from the key secreted precursor. Human axillary odor is formed by the action of Corynebacteria on odorless axilla secretions. Sulfanylalkanols, 3-methyl-3-sulfanylhexan-1-ol in particular, form one key class of the odoriferous compounds. A conjugate with the dipeptide Cys-Gly has been reported as the secreted precursor for 3-methyl-3-sulfanylhexan-1-ol. Here, we confirm the Cys-Gly-(S) conjugate as the major precursor of this odorant, with lower levels of the Cys-(S) conjugate being present in axilla secretions. The enzymatic release of 3-methyl-3-sulfanylhexan-1-ol from the Cys-Gly-(S) conjugate by the axilla isolate Corynebacterium Ax20 was thus investigated. Cellular extracts of Ax20 released 3-methyl-3-sulfanylhexan-1-ol from the Cys-Gly-(S) conjugate and from the Cys-(S) conjugate, whereas the previously isolated C-S lyase of this bacterial strain was only able to cleave the Cys-(S) conjugate. o-Phenanthroline blocked the release from the Cys-Gly-(S) conjugate but did not affect cleavage of the Cys-(S) conjugate, indicating that in a first step, a metal-dependent dipeptidase hydrolyzes the Cys-Gly bond. This enzyme was purified by four chromatographic steps and gel electrophoresis, and the partial amino acid sequence was determined. The corresponding gene was cloned and expressed in Escherichia coli. It codes for a novel dipeptidase with a high affinity toward the Cys-Gly-(S) conjugate of 3-methyl-3-sulfanylhexan-1-ol. Co-incubating either the synthetic Cys-Gly-(S) conjugate or fresh axilla secretions with both the C-S lyase and the novel dipeptidase did release 3-methyl-3-sulfanylhexan-1-ol, proving that the sequential action of these two enzymes from the skin bacterium Corynebacterium Ax20 does release the odorant from the key secreted precursor. The skin in human armpits contains a dense arrangement of sweat glands. Volatile substances evaporating from these areas make a key contribution to human body odor. However, sweat secreted from apocrine glands in these skin areas is initially odorless, and since the work of Shelley et al. (1Shelley W.B. Hurley H.J. Nichols A.C. Arch. Dermatol. Syphilol. 1953; 68: 430-446Crossref Scopus (128) Google Scholar), it is known that skin bacteria release the odoriferous principles from non-smelling substrates present in these secretions. Indeed, the axilla is a skin region colonized by an unusually dense bacterial population, with a species composition dominated by the two genera Staphylococcus and Corynebacterium (2Leyden J.J. McGinley K.J. Hoelzle E. Labows J.N. Kligman A.M. J. Investig. Dermatol. 1981; 77: 413-416Abstract Full Text PDF PubMed Scopus (295) Google Scholar, 3Shehadeh N. Kligman A. J. Investig. Dermatol. 1963; 41: 1-5Abstract Full Text PDF PubMed Google Scholar). Most individuals carry a flora that is dominated by either one of these two genera, and there is a strong correlation between a high population of Corynebacteria and strong axillary odor formation (2Leyden J.J. McGinley K.J. Hoelzle E. Labows J.N. Kligman A.M. J. Investig. Dermatol. 1981; 77: 413-416Abstract Full Text PDF PubMed Scopus (295) Google Scholar, 4Jackman P.J. Noble W.C. Clin. Exp. Dermatol. 1983; 8: 259-268Crossref PubMed Scopus (56) Google Scholar). Subjects whose axillary skin is mainly colonized by Staphylococci emit only low levels of odor. Based on this fundamental work, axilla secretions contain non-odoriferous precursors that are transformed into the volatile substances by bacterial enzymes mainly present in Corynebacteria and to a lesser extent in Staphylococci. Early studies on the chemistry of human axilla odors identified the odoriferous steroids 5α-androst-16-en-3-one (5Claus R. Alsing W. J. Endocrinol. 1976; 68: 483-484Crossref PubMed Scopus (83) Google Scholar, 6Bird S. Gower D.B. J. Steroid Biochem. 1981; 14: 213-219Crossref PubMed Scopus (47) Google Scholar) and 5α-androst-16-en-3α-ol (7Brooksbank B.W.L. Brown R. Gustafsson J.A. Experientia (Basel). 1974; 30: 864-865Crossref PubMed Scopus (86) Google Scholar) in human axilla secretions. Later, Zeng et al. (8Zeng X.N. Leyden J.J. Lawley H.J. Sawano K. Nohara I. Preti G. J. Chem. Ecol. 1991; 17: 1469-1492Crossref PubMed Scopus (216) Google Scholar) reported that short, branched fatty acids make a major contribution to the axilla odor with (E)-3-methyl-2-hexenoic acid being the key component. In our previous work, we had shown that a broad diversity of other unsaturated or hydroxylated odorant acids is present in hydrolyzed axilla secretions and that all these acids are released from the glands in the form of odorless glutamine conjugates (9Natsch A. Gfeller H. Gygax P. Schmid J. Acuña G. J. Biol. Chem. 2003; 278: 5718-5727Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 10Natsch A. Derrer S. Flachsmann F. Schmid J. Chemistry & Biodiversity. 2006; 3: 1-20Crossref PubMed Scopus (118) Google Scholar). We had isolated a specific zinc-dependent aminoacylase from the axilla isolate Corynebacterium striatum Ax20, which catalyzes the release of the odoriferous principles from these glutamine conjugates (9Natsch A. Gfeller H. Gygax P. Schmid J. Acuña G. J. Biol. Chem. 2003; 278: 5718-5727Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The third and most recently discovered class of human axilla odorants are volatile sulfanylalkanols (11Troccaz M. Starkenmann C. Niclass Y. van de Waal M. Clark A.J. Chemistry & Biodiversity. 2004; 1: 1022-1035Crossref PubMed Scopus (88) Google Scholar, 12Natsch A. Schmid J. Flachsmann F. Chemistry & Biodiversity. 2004; 1: 1058-1072Crossref PubMed Scopus (97) Google Scholar, 13Hasegawa Y. Yabuki M. Matsukane M. Chemistry & Biodiversity. 2004; 1: 2042-2050Crossref PubMed Scopus (79) Google Scholar), with 3-methyl-3-sulfanylhexan-1-ol (3M3SH) 2The abbreviations used are: 3M3SH, 3-methyl-3-sulfanylhexan-1-ol; GC-FPD, gas-chromatography with flame photometric detection; LC, liquid chromatography; MS, mass spectrometry; MS2, two-stage mass analysis; HPLC, high pressure liquid chromatography; Ni-NTA, nickel-nitrilotriacetic acid. 2The abbreviations used are: 3M3SH, 3-methyl-3-sulfanylhexan-1-ol; GC-FPD, gas-chromatography with flame photometric detection; LC, liquid chromatography; MS, mass spectrometry; MS2, two-stage mass analysis; HPLC, high pressure liquid chromatography; Ni-NTA, nickel-nitrilotriacetic acid. as the quantitatively dominating compound within this structural class. This compound can be released both from a Cys-(S) conjugate and from axilla secretions by a C-S lyase cloned from Corynebacterium Ax20, indicating that Cys-(S) conjugates could be physiological precursors for this compound class (12Natsch A. Schmid J. Flachsmann F. Chemistry & Biodiversity. 2004; 1: 1058-1072Crossref PubMed Scopus (97) Google Scholar). However, it was later shown that a Cys-Gly-(S) conjugate of 3M3SH is secreted by human subjects (14Starkenmann C. Niclass Y. Troccaz M. Clark A.J. Chemistry & Biodiversity. 2005; 2: 705-716Crossref PubMed Scopus (65) Google Scholar) (for structures, see Fig. 1) and that bacterial cultures of Staphylococcus haemolyticus can release 3M3SH from this dipeptide precursor. In a recent patent application, this activity was attributed to a β-lyase, but the corresponding enzyme was neither isolated from S. haemolyticus, nor has it been characterized (15Starkenmann C. Clark A. Troccaz M. Niclass Y. PCT International Patent Application WO 2006/079934 A2. March 8, 2006; Google Scholar). Thus, the enzymatic release of the key axilla odorant 3M3SH by skin bacteria from the physiological dipeptide precursor has not yet been deciphered. Here, we report the isolation and the characterization of a novel specific dipeptidase and the corresponding gene from the axilla isolate Corynebacterium Ax20. We show that the secreted Cys-Gly-(S) conjugate of 3M3SH first needs to be cleaved by this dipeptidase and only afterward becomes a substrate of the previously reported C-S lyase, which finally releases 3M3SH. Materials—Unless otherwise noted, all chemicals were purchased from Fluka (Buchs, Switzerland). (Z)-protected amino acids and peptides as enzyme substrates were from Aldrich (Buchs, Switzerland) and from Senn Chemicals (Dielsdorf, Switzerland). All columns and chromatography resins were from Amersham Biosciences (Otelfingen, Switzerland) with the exception of Ni-NTA agarose purchased from Qiagen (Hombrechtikon, Switzerland). (S)-(1-(2-hydroxyethyl)-1-methylbutyl)-l-cysteinylglycine (Cys-Gly-(S) conjugate) was synthesized by the method described by Starkenmann et al. (14Starkenmann C. Niclass Y. Troccaz M. Clark A.J. Chemistry & Biodiversity. 2005; 2: 705-716Crossref PubMed Scopus (65) Google Scholar). 3M3SH, its Cys-(S) conjugate, and Gln conjugates of carboxylic acids as reference substrates were synthesized as described before (9Natsch A. Gfeller H. Gygax P. Schmid J. Acuña G. J. Biol. Chem. 2003; 278: 5718-5727Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 10Natsch A. Derrer S. Flachsmann F. Schmid J. Chemistry & Biodiversity. 2006; 3: 1-20Crossref PubMed Scopus (118) Google Scholar, 12Natsch A. Schmid J. Flachsmann F. Chemistry & Biodiversity. 2004; 1: 1058-1072Crossref PubMed Scopus (97) Google Scholar). The recombinant C-S lyase from Corynebacterium Ax20 was expressed and purified on a Ni-NTA-affinity column (12Natsch A. Schmid J. Flachsmann F. Chemistry & Biodiversity. 2004; 1: 1058-1072Crossref PubMed Scopus (97) Google Scholar). Axilla secretions of individual donors were sampled on cotton pads fixed in the axillary region during physical exercise (10Natsch A. Derrer S. Flachsmann F. Schmid J. Chemistry & Biodiversity. 2006; 3: 1-20Crossref PubMed Scopus (118) Google Scholar). Bacterial Strains—Isolation and characterization of axilla bacteria were described previously (9Natsch A. Gfeller H. Gygax P. Schmid J. Acuña G. J. Biol. Chem. 2003; 278: 5718-5727Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The bacterial strains were grown on tryptic soy agar supplemented with 0.01% Tween 80 as lipid source. For enzyme purification or enzyme assays, axilla bacteria were grown in Mueller-Hinton broth supplemented with 0.01% Tween 80. E. coli strain TOP10, used for the expression of recombinant enzymes, was grown in LB broth. LC-MS Analysis of Axilla Secretions—The aqueous fraction of axilla secretions was fractionated over a Superdex peptide 10/300 GL column using (NH4)2CO3 (100 mm) as elution buffer, and the single fractions were analyzed with LC-MS with the method laid out in Natsch et al. (10Natsch A. Derrer S. Flachsmann F. Schmid J. Chemistry & Biodiversity. 2006; 3: 1-20Crossref PubMed Scopus (118) Google Scholar). In brief, a Finnigan LCQ mass spectrometer operated in the atmospheric pressure chemical ionization mass spectrometry mode and equipped with a Flux Rheos 2000 HPLC pump was used, and HPLC separation was performed on a C18 RP column modified for proteins and peptides (Grace Vydac, Hesperia, CA). The mobile phase consisted of H2O (A) and MeOH (B) each containing 1% HOAc (v/v). GC-FPD Analysis for Release of 3M3SH—The Cys- and Cys-Gly-(S) conjugates of 3M3SH or the aqueous fraction of axilla secretions were incubated with the recombinant enzymes or bacterial extracts for 2 h. The aqueous phase (500 μl) was extracted with 250 μl of methyl-tert-butyl-ether, and 6 μl were injected in the splitless pulse-pressure mode onto a SPW1-sulfur column (Supelco, Bellefonte, PA) mounted on an Agilent GC 6890N (Agilent, Wilmington, DE) system with a flame photometric detector specific for sulfur chemicals. The temperature program was set to 2 min of initial temperature at 50 °C, heating at a rate of 10 °C/min to 240 °C, and a final 15 min at 240 °C. Dipeptidase Activity Assay—Bacterial extracts, column fractions, or the purified peptidase were incubated with the Cys-Gly-(S) conjugate or (S)-benzyl-Cys-Gly at a final concentration of 0.5 or 1 mm and with an excess of β-lyase (10 μg/ml) in buffer A (50 mm NaCl, 50 mm NaH2PO4/K2HPO4, pH 7) in a final volume of 100 μl. Release of 3M3SH or benzylthiol was detected by adding 50 μl of a 1 mm solution of the thiol-specific fluorescent probe monobromobimane dissolved in NaCO3 buffer (100 mm, pH 8.8) and, after a 5-min reaction time, fluorescence measurement on a Flexstation (Molecular Devices, Sunnyvale, CA) with an excitation at 390 nm and emission at 478 nm. To determine enzyme kinetics, the same assay was used, but the enzyme reaction with the dipeptidase was stopped by adding EDTA (0.1 mm), and only then the β-lyase was added to release the thiol for fluorescent detection. Thin Layer Chromatography for Peptide Cleavage—Cys- and Cys-Gly-(S) conjugates, dipeptides and acetyl ornithine (1 mm solutions) were incubated with the peptidase for 1 h. 10 μl of each reaction were spotted on TLC plates and developed with a mixture of 1-butanol, acetic acid, H2O (4:1:1) in the case of the Cys- and Cys-Gly-(S) conjugates or with H2O and 1-propanol (1:1) in the case of the dipeptides. Products were visualized by spraying a ninhydrin solution and heating the plates until the completion of the ninhydrin reaction. Protein Determination and SDS-PAGE—Protein concentrations were determined with the Bradford reagent (Bio-Rad) using bovine serum albumin as standard. SDS-PAGE was performed according to Laemmli (16Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206971) Google Scholar) with 5% stacking gels and 10% separation gels. Protein bands were visualized by Coomassie Blue staining. Purification of the Peptidase—C. striatum Ax20 was selected for the purification of the enzyme responsible for the cleavage of the Cys-Gly-(S) conjugate. It was grown for 42 h, and cells from a total culture volume of four liters were washed once with buffer A and resuspended in a final volume of 5 ml of buffer A. Using glass beads (425–600 μm, Sigma), the cells were mechanically disrupted by vortexing them at maximal speed for 20 min. The lysate was cleared by centrifugation and passed through a 0.45-μm syringe filter. The extract was then sequentially run over four chromatography columns: 1) phenyl-sepharose hydrophobic interaction resin, elution with a linear gradient from 1000 to 0 mm (NH4)2SO4 in Buffer A; 2) Mono Q strong anion exchange column on the fast protein liquid chromatography system, elution with a gradient from 0 to 800 mm KCl in buffer A; 3) Mono P weak anion exchange column on the fast protein liquid chromatography system, elution with a gradient from 0 to 800 mm KCl in 50 mm bis-Tris buffer (pH 6.5); 4) Superdex 200 gel filtration column, elution with buffer A. After each column separation, active fractions (determined by the fluorescent assay) were pooled and desalted by dilution/ultrafiltration. Fractions of the final Superdex gel filtration step were run on a 10% SDS-PAGE gel. The single protein band common to the active fractions was isolated and subjected to tryptic digestion, and the sequence of internal peptides was determined with LC-electrospray mass ionization-tandem MS analysis (Genomic Center, University of Zürich, Switzerland). Molecular Biology Methods—Chromosomal DNA of Ax20 was obtained from cell lysates by proteinase K digestion and subsequent extraction with cetyltrimethylammonium bromide/NaCl and chloroform/isoamylacetate (17Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, New York1995Google Scholar). Based on the partial amino acid sequences of the purified enzyme, degenerated primers were designed to amplify genomic DNA fragments. Standard PCR conditions were used according to the manufacturer (Taq polymerase, Sigma, Buchs, Switzerland). The amplified DNA was submitted to Microsynth (Balgach, Switzerland) for sequence analysis. Based on the obtained partial sequence, specific nested oligonucleotides were designed to clone the upstream and downstream regions. Chromosomal DNA of Ax20 was digested with SmaI and PvuII and ligated to the GenomeWalker Adaptor (Clontech). The upstream and downstream regions were then amplified as described in the instructions to the Universal GenomeWalker™ kit (Clontech Laboratories) and sequenced. The resulting open reading frame was amplified from chromosomal DNA of Ax20 by PCR using the specific primers 5′-CGA CAT GCC ATG GGC AGC AAC GAC AAG GCA GCA ACC AGC-3′ and 5′-CGA CAT AAG CTT TTT CCC GTA GGT GAG CAG GAA T-3′. The amplified DNA fragment was digested with NcoI and HindIII and ligated into the vector pBAD/myc-HisA (Invitrogen, Groningen, The Netherlands) predigested with the same enzymes. The resulting plasmid pBAD/mycHis-tpdA was transformed into the host strain E. coli TOP10 (Invitrogen). This strain was grown in LB broth until it reached an A600 of 0.5. The culture was supplemented with arabinose (0.2% final concentration) to induce gene expression, further incubated for 4 h, and harvested by centrifugation, and the cells were disrupted using a French Press. The His-tagged recombinant peptidase was finally purified using a Ni-NTA column according to the manufacturer's instructions. LC-MS Analysis of Axilla Fractions to Detect Amino Acid Conjugates of 3M3SH—Aqueous fractions of axilla secretions obtained by gel filtration were analyzed by LC-MS in comparison with synthetic Cys- and Cys-Gly-(S) conjugates of 3M3SH. In axilla secretions, a significant peak at retention time 4.85 min was observed on the extracted mass trace m/z 293, corresponding to the protonated Cys-Gly-(S) conjugate (Fig. 2, A-2 and B-2). This peak can only be detected in axilla secretions on the selected ion trace, whereas the total ion current (TIC) trace contains two dominant peaks (Fig. 2, B-1), which correspond to the two key Gln conjugates of carboxylic acids reported before (9Natsch A. Gfeller H. Gygax P. Schmid J. Acuña G. J. Biol. Chem. 2003; 278: 5718-5727Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), as verified based on MS analysis and comparison with synthetic references (data not shown). The Cys-(S) conjugate of 3M3SH yields an 8-fold lower signal intensity in LC-MS analysis as compared with the Cys-Gly-(S) conjugate (compare the different normalization level NL in Fig. 2, A-2 and A-3), probably due to less efficient ionization of the Cys-(S) conjugate. In axilla secretions, on the m/z trace 236, only a very minor peak at the correct retention time was observed, putatively corresponding to the Cys-(S) conjugate (indicated in Fig. 2, B-3 by an arrow). Indeed, the on-line MS2 spectrum of this peak showed the same base ion at m/z 122 as the MS2 spectrum of the synthetic reference sample. This ion presumably corresponds to the protonated free Cys (supplemental Fig. S1). Nevertheless, even considering the lower response factor for the Cys-(S) conjugate, this compound, albeit clearly present, is the minor component, and thus, the Cys-Gly-(S) conjugate of 3M3SH indeed is the main precursor present in axilla secretions. 3M3SH Is Released from the Cys-Gly-(S) Conjugate by the Sequential Action of a Metallopeptidase and a β-Lyase—The fact that mainly the Cys-Gly-(S) conjugate and only small amounts of the Cys-(S) conjugate can be found in fresh sweat suggests that 3M3SH is directly released either by a β-lyase in axilla bacteria from the Cys-Gly-(S) conjugate or from a Cys-(S) conjugate that has been generated from the Cys-Gly-(S) conjugate by hydrolysis of the Cys-Gly peptide bond. To test the two hypotheses, the synthetic Cys- and the Cys-Gly-(S) conjugates of 3M3SH were incubated with the purified β-lyase from the axilla strain Ax20 or with total cell extracts of several bacterial strains isolated from the axilla. As shown in Fig. 3, the purified β-lyase and the Ax20 extract released significant amounts of 3M3SH from the Cys-(S) conjugate, and this could not be blocked by the metallopeptidase inhibitor o-phenanthroline. When incubated with the Cys-Gly-(S) conjugate, only the Ax20 extract but not the recombinant β-lyase released a significant amount of 3M3SH. Interestingly, this reaction could be blocked by o-phenanthroline, suggesting that the strain Ax20 harbors an o-phenanthroline-sensitive dipeptidase that hydrolyzes the Cys-Gly-(S) conjugate, thereby producing the substrate for the β-lyase. Extracts of Staphylococcus and Micrococcus strains did not cleave either of the two conjugates, whereas other tested Corynebacteria showed only weak β-lyase activity (Fig. 3). Corynebacterium jeikeium K411 is the only axilla isolate whose genome had been sequenced (19Tauch A. Kaiser O. Hain T. Goesmann A. Weisshaar B. Albersmeier A. Bekel T. Bischoff N. Brune I. Chakraborty T. Kalinowski J. Meyer F. Rupp O. Schneiker S. Viehoever P. Puehler A. J. Bacteriol. 2005; 187: 4671-4682Crossref PubMed Scopus (157) Google Scholar). After a 2-h incubation, no significant cleavage of the Cys-Gly-(S) conjugate by this strain was observed; however, after prolonged incubation it slowly cleaved this substrate (data not shown). The structures of the conjugates along with these proposed reactions are shown in Fig. 1. Purification of the Novel Metallopeptidase and Cloning of the Corresponding Gene—The unknown metallopeptidase cleaving the Cys-Gly-(S) conjugates of 3M3SH was then purified from cellular extracts of C. striatum Ax20 by activity-guided fractionation as described under "Experimental Procedures." Two columns were tested for the first purification step, a Mono-Q and a phenyl-Sepharose column. From both columns, the metallopeptidase activity eluted as a single peak. Also, from all subsequent column fractionations, only a single peak of activity was recovered, indicating that only one key enzyme is involved in the hydrolysis of the Cys-Gly-(S) conjugate. Fractions of the last purification step were analyzed by SDS-PAGE. Three different polypeptides were left in the active fractions. Comparison of the relative peptidase activity and the intensity of the bands resulted in a clear candidate protein, which had an apparent mass of ∼50 kDa (see supplemental Fig. S2 in the supporting information). The candidate protein was excised and submitted to a tryptic digest and sequence analysis, leading to the amino acid sequence of several internal peptides. A data base search with the obtained peptide sequences revealed homology to putative peptidases, suggesting that the analyzed protein could indeed be responsible for the cleavage of the Cys-Gly bond. Based on four peptide sequences, degenerated primers were designed, and a total of 20 primer combinations were used for PCR amplification with chromosomal DNA of Ax20 as template. Two primer combinations led to specific products of 281 and 272 bp, respectively. The obtained PCR products were sequenced, and based on these partial sequences, oligonucleotides were designed to clone the upstream and downstream regions by genome walking using libraries generated from PvuII and SmaI digests of chromosomal Ax20 DNA. An upstream fragment of 600 bp and a downstream fragment of 2400 bp were obtained. Within these sequenced regions, open reading frames coding for the N-terminal part and the C-terminal part were identified. Finally, the complete open reading frame was amplified by PCR using Ax20 genomic DNA as template, and it was cloned into the bacterial expression vector pBAD/mycHisA. The sequence was deposited in the GenBank™ under accession number EU311559. The gene was named tpdA, which stands for "thiol precursor dipeptidase." Sequence Comparison with Related Proteins—The protein deduced from the open reading frame has a high homology to a large number of putative bacterial peptidases belonging to the M20 family of metallopeptidases. Closely related genes, for which no function has been identified yet, exist in the genomes of most other members of the class of actinobacteria. The closest homologues in E. coli are the succinyl-diaminopimelate desuccinylase and the acetyl-ornithine deacetylase (18Boyen A. Charlier D. Charlier J. Sakanyan V. Mett I. Glansdorff N. Gene (Amst.). 1992; 116: 1-6Crossref PubMed Scopus (38) Google Scholar), two related proteins also belonging to the M20 family of peptidases, which are involved in the biosynthesis of lysine and arginine, respectively. The sequence alignment of the dipeptidase with these genes of known function, with the closest relatives from Corynebacterium diphteriae and C. jeikeium K411 (19Tauch A. Kaiser O. Hain T. Goesmann A. Weisshaar B. Albersmeier A. Bekel T. Bischoff N. Brune I. Chakraborty T. Kalinowski J. Meyer F. Rupp O. Schneiker S. Viehoever P. Puehler A. J. Bacteriol. 2005; 187: 4671-4682Crossref PubMed Scopus (157) Google Scholar) and with the carboxypeptidase G2 from Pseudomonas aeruginosa (18Boyen A. Charlier D. Charlier J. Sakanyan V. Mett I. Glansdorff N. Gene (Amst.). 1992; 116: 1-6Crossref PubMed Scopus (38) Google Scholar, 20Minton N.P. Atkinson T. Bruton C.J. Sherwood R.F. Gene (Amst.). 1984; 31: 31-38Crossref PubMed Scopus (70) Google Scholar), is shown in supplemental Fig. S3 in the supporting information. Characterization of the Pure Recombinant Enzyme—Transformants of E. coli strain TOP10 harboring the plasmid pBAD/mycHis-tpdA expressed high levels of the His-tagged recombinant protein, which was purified to >95% purity using a Ni-NTA column. To demonstrate that the isolated protein indeed catalyzes the hydrolysis of the peptide bond of the Cys-Gly-(S) conjugate, this substrate was incubated with the purified protein, and the reaction products were separated by TLC. As shown in Fig. 4, the Cys-Gly-(S) conjugate was indeed hydrolyzed to the Cys-(S) conjugate and glycine, and the same reaction was performed by the TpdA with (S)-benzyl-Cys-Gly as a substrate (data not shown). Having shown this, we tried to find additional substrates for the identified enzyme. A series of 24 dipeptides was incubated for 1 h with the peptidase and then analyzed by TLC. The peptidase did hydrolyze a relatively broad range of dipeptides, but it was not able to cleave acidic amino acids from the C terminus (Table 1). In addition, the peptidase did not hydrolyze dipeptides with glycine in the second position unless a bulky hydrophobic residue was present in the N-terminal position. Thus, although (S)-substituted Cys-Gly conjugates are very good substrates, unsubstituted Cys-Gly is not cleaved.TABLE 1Substrate specificity of TpdA and the native enzyme preparationSubstrateRecombinant TpdANative enzyme preparationb1 mm substrate was incubated with different dilutions of the native enzymatic preparation obtained after the fourth column (Superdex gel filtration). - indicates no hydrolysis; +/- indicates traces of product detected with 1:1 dilution; + indicates >20% of substrate hydrolyzed with 1:1 dilution; ++ indicates >20% of substrate hydrolyzed with 1:3 dilution; +++ indicates >20% of substrate hydrolyzed with 1:10 dilution; ++++ indicates >20% of substrate hydrolyzed with 1:30 dilution. The native preparation used had an apparent purity of 30%. ND, not determined.Dipeptides and derivatives (S) 3M3SH-Cys-Gly++++ (S) Benzyl-Cys-Gly++++++ Cys-Gly-- Gly-Gly-ND Lys-Gly-ND Phe-Gly++++ Trp-Leu++++++ Glu-Trp+++++ Ile-Asn+++ND Leu-Ala+++ND Leu-Asn+++ND Val-Lys+++ND Ala-Ala++++ Ser-Leu++ND Thr-Gln++ND Val-Gln++ND Ile-Leu++ Trp-Val+ND Ala-Glu-- Arg-Glu-ND Asp-Asp-- Asp-Glu-ND Glu-Asp-ND Glu-Glu-- Ile-Trp-- Ile-Val-ND Val-Glu-NDAcyl-amino acidsND Acetyl-ornithine-ND Nα-Lauroyl-Gln+ND Nα-(E)-3-methyl-2-hexenoyl-glutamine+/-ND Nα-3-methyl-3-hydroxy-hexanoyl-glutamine+/-NDa 1 mm substrate was incubated with different concentrations of TpdA for 1 h. - indicates no hydrolysis; +/- indicates traces of product detected with 1 μg/ml enzyme; >20% of substrate hydrolyzed with 1 μg/ml enzyme; ++ indicates >20% of substrate hydrolyzed with 0.3 μg/ml enzyme; +++ indicates >20% of substrate hydrolyzed with 0.1 μg/ml enzyme; ++++ indicates >20% of substrate hydrolyzed with 0.03 μg/ml enzyme.b 1 mm substrate was incubated with different dilutions of the native enzymatic preparation obtained after the fourth column (Superdex gel filtration). - indicates no hydrolysis; +/- indicates traces of product detected with 1:1 dilut
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