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• W2079781629 abstract "Homocysteine (Hcy) editing by methionyl-tRNA synthetase results in the formation of Hcy-thiolactone and initiates a pathway that has been implicated in human disease. In addition to being cleared from the circulation by urinary excretion, Hcy-thiolactone is detoxified by the serum Hcy-thiolactonase/paraoxonase carried on high density lipoprotein. Whether Hcy-thiolactone is detoxified inside cells was unknown. Here we show that Hcy-thiolactone is hydrolyzed by an intracellular enzyme, which we have purified to homogeneity from human placenta and identified by proteomic analyses as human bleomycin hydrolase (hBLH). We have also purified an Hcy-thiolactonase from the yeast Saccharomyces cerevisiae and identified it as yeast bleomycin hydrolase (yBLH). BLH belongs to a family of evolutionarily conserved cysteine aminopeptidases, and its only known biologically relevant function was deamidation of the anticancer drug bleomycin. Recombinant hBLH or yBLH, expressed in Escherichia coli, exhibits Hcy-thiolactonase activity similar to that of the native enzymes. Active site mutations, C73A for hBLH and H369A for yBLH, inactivate Hcy-thiolactonase activities. Yeast blh1 mutants are deficient in Hcy-thiolactonase activity in vitro and in vivo, produce more Hcy-thiolactone, and exhibit greater sensitivity to Hcy toxicity than wild type yeast cells. Our data suggest that BLH protects cells against Hcy toxicity by hydrolyzing intracellular Hcy-thiolactone. Homocysteine (Hcy) editing by methionyl-tRNA synthetase results in the formation of Hcy-thiolactone and initiates a pathway that has been implicated in human disease. In addition to being cleared from the circulation by urinary excretion, Hcy-thiolactone is detoxified by the serum Hcy-thiolactonase/paraoxonase carried on high density lipoprotein. Whether Hcy-thiolactone is detoxified inside cells was unknown. Here we show that Hcy-thiolactone is hydrolyzed by an intracellular enzyme, which we have purified to homogeneity from human placenta and identified by proteomic analyses as human bleomycin hydrolase (hBLH). We have also purified an Hcy-thiolactonase from the yeast Saccharomyces cerevisiae and identified it as yeast bleomycin hydrolase (yBLH). BLH belongs to a family of evolutionarily conserved cysteine aminopeptidases, and its only known biologically relevant function was deamidation of the anticancer drug bleomycin. Recombinant hBLH or yBLH, expressed in Escherichia coli, exhibits Hcy-thiolactonase activity similar to that of the native enzymes. Active site mutations, C73A for hBLH and H369A for yBLH, inactivate Hcy-thiolactonase activities. Yeast blh1 mutants are deficient in Hcy-thiolactonase activity in vitro and in vivo, produce more Hcy-thiolactone, and exhibit greater sensitivity to Hcy toxicity than wild type yeast cells. Our data suggest that BLH protects cells against Hcy toxicity by hydrolyzing intracellular Hcy-thiolactone. Homocysteine (Hcy) 2The abbreviations and trivial name used are: Hcy, homocysteine; AMC, aminomethylcoumarine; R-AMC, arginine aminomethylcoumarylamide; BLH, bleomycin hydrolase; hBLH, human BLH; yBLH, yeast BLH; E-64, trans-epoxysuccinyl-l-leucilamido-(4-guanidino)butane; HPLC, high performance liquid chromatography; HTLase, homocysteine-thiolactonase; hHTLase, human HTLase; yHTLase, yeast HTLase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight.2The abbreviations and trivial name used are: Hcy, homocysteine; AMC, aminomethylcoumarine; R-AMC, arginine aminomethylcoumarylamide; BLH, bleomycin hydrolase; hBLH, human BLH; yBLH, yeast BLH; E-64, trans-epoxysuccinyl-l-leucilamido-(4-guanidino)butane; HPLC, high performance liquid chromatography; HTLase, homocysteine-thiolactonase; hHTLase, human HTLase; yHTLase, yeast HTLase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. is a sulfur-containing amino acid that is found as a normal metabolite in the three domains of life. In all organisms, Hcy is metabolized to Hcy-thiolactone by methionyl-tRNA synthetase in an error-editing reaction in protein biosynthesis when Hcy becomes mistakenly selected in place of methionine (reviewed in Refs. 1Jakubowski H. Cell. Mol. Life Sci. 2004; 61: 470-487Crossref PubMed Scopus (210) Google Scholar, 2Jakubowski H. Ibba M. Cusack S. Francklyn C. The Aminoacyl-tRNA Synthetases. Landes Biosciences, Georgetown, TX2005: 384-396Google Scholar, 3Jakubowski H. J. Nutr. 2006; 136: 1741S-1749SCrossref PubMed Google Scholar). In each organism examined (bacteria, yeast, plant, mouse, and human) the Hcy-thiolactone pathway becomes predominant when remethylation or trans-sulfuration reactions are impaired by genetic alterations of enzymes involved in Hcy metabolism, such as cystathionine β-synthase (4Jakubowski H. EMBO J. 1991; 10: 593-598Crossref PubMed Scopus (70) Google Scholar, 5Jakubowski H. J. Biol. Chem. 1997; 272: 1935-1941Abstract Full Text Full Text PDF PubMed Google Scholar, 6Jakubowski H. Anal. Biochem. 2002; 308: 112-119Crossref PubMed Scopus (79) Google Scholar) and methionine synthase (4Jakubowski H. EMBO J. 1991; 10: 593-598Crossref PubMed Scopus (70) Google Scholar, 6Jakubowski H. Anal. Biochem. 2002; 308: 112-119Crossref PubMed Scopus (79) Google Scholar), or by inadequate supply of folate (5Jakubowski H. J. Biol. Chem. 1997; 272: 1935-1941Abstract Full Text Full Text PDF PubMed Google Scholar, 7Jakubowski H. Zhang L. Bardeguez A. Aviv A. Circ. Res. 2000; 87: 45-51Crossref PubMed Scopus (263) Google Scholar, 8Senger B. Despons L. Walter P. Jakubowski H. Fasiolo F. J. Mol. Biol. 2001; 311: 205-216Crossref PubMed Scopus (20) Google Scholar, 9Jakubowski H. Guranowski A. J. Biol. Chem. 2003; 278: 6765-6770Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), vitamin B12, or vitamin B6.In recent years, Hcy has become a focus of intense studies in the context of human pathophysiology. Elevated serum Hcy levels observed in genetic disorders of Hcy metabolism are associated with severe pathologies, which affect multiple organs and lead to premature death due to vascular complications (10Lentz S.R. Thromb. Haemost. 2005; 3: 1646-1654Crossref Scopus (306) Google Scholar). Although severe hyperhomocysteinemia is rare, mild hyperhomocysteinemia is quite prevalent in a general population and is associated with an increased risk of cardiovascular (11Collaboration Homocysteine Studies J. Am. Med. Assoc. 2002; 288: 2015-2022Crossref PubMed Scopus (1901) Google Scholar) and neurodegenerative diseases, such as Alzheimer disease (12Seshadri S. Beiser A. Selhub J. Jacques P.F. Rosenberg I.H. D'Agostino R.B. Wilson P.W. Wolf P.A. N. Engl. J. Med. 2002; 346: 476-483Crossref PubMed Scopus (2804) Google Scholar). The strongest evidence that Hcy plays a causal role in cardiovascular disease comes from studies of hyperhomocysteinemia in animal models (10Lentz S.R. Thromb. Haemost. 2005; 3: 1646-1654Crossref Scopus (306) Google Scholar) and small trials in humans (13Loscalzo J. N. Engl. J. Med. 2006; 354: 1629-1632Crossref PubMed Scopus (260) Google Scholar). Although large clinical trials testing whether lowering Hcy can lead to better vascular outcomes have not been successful (13Loscalzo J. N. Engl. J. Med. 2006; 354: 1629-1632Crossref PubMed Scopus (260) Google Scholar), an efficacy analysis shows that high risk stroke patients do benefit from lowering of plasma Hcy by vitamin supplementation (14Spence J.D. Bang H. Chambless L.E. Stampfer M.J. Stroke. 2005; 36: 2404-2409Crossref PubMed Scopus (230) Google Scholar).Although Hcy is a normal metabolite, its excess can be extremely toxic to human (15Zhang C. Cai Y. Adachi M.T. Oshiro S. Aso T. Kaufman R.J. Kitajima S. J. Biol. Chem. 2001; 276: 35867-35874Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 16Hossain G.S. van Thienen J.V. Werstuck G.H. Zhou J. Sood S.K. Dickhout J.G. De Koning A.B. Tang D. Wu D. Falk E. Poddar R. Jacobsen D.W. Zhang K. Kaufman R.J. Austin R.C. J. Biol. Chem. 2003; 278: 30317-30327Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 17Roybal C.N. Yang S. Sun C.W. Hurtado D. van der Jagt D.L. Townes T.M. Abcouwer S.F. J. Biol. Chem. 2004; 279: 14844-14852Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), animal (18Mattson M.P. Shea T.B. Trends Neurosci. 2003; 26: 137-146Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar), yeast (4Jakubowski H. EMBO J. 1991; 10: 593-598Crossref PubMed Scopus (70) Google Scholar, 6Jakubowski H. Anal. Biochem. 2002; 308: 112-119Crossref PubMed Scopus (79) Google Scholar, 19Jakubowski H. Goldman E. Microbiol. Rev. 1992; 56: 412-429Crossref PubMed Google Scholar), and bacterial cells (20Tuite N.L. Fraser K.R. O'Byrne C.P. J. Bacteriol. 2005; 187: 4362-4371Crossref PubMed Scopus (43) Google Scholar). Why Hcy is toxic is not entirely clear and is a subject of intense studies (1Jakubowski H. Cell. Mol. Life Sci. 2004; 61: 470-487Crossref PubMed Scopus (210) Google Scholar, 3Jakubowski H. J. Nutr. 2006; 136: 1741S-1749SCrossref PubMed Google Scholar, 10Lentz S.R. Thromb. Haemost. 2005; 3: 1646-1654Crossref Scopus (306) Google Scholar). One hypothesis suggests that the conversion to Hcy-thiolactone contributes to Hcy toxicity and is linked to atherosclerosis in humans (1Jakubowski H. Cell. Mol. Life Sci. 2004; 61: 470-487Crossref PubMed Scopus (210) Google Scholar, 3Jakubowski H. J. Nutr. 2006; 136: 1741S-1749SCrossref PubMed Google Scholar, 5Jakubowski H. J. Biol. Chem. 1997; 272: 1935-1941Abstract Full Text Full Text PDF PubMed Google Scholar, 7Jakubowski H. Zhang L. Bardeguez A. Aviv A. Circ. Res. 2000; 87: 45-51Crossref PubMed Scopus (263) Google Scholar). The formation of Hcy-thiolactone can be detrimental for two reasons. First, it requires ATP and thus causes nonproductive consumption of cellular energy (4Jakubowski H. EMBO J. 1991; 10: 593-598Crossref PubMed Scopus (70) Google Scholar, 19Jakubowski H. Goldman E. Microbiol. Rev. 1992; 56: 412-429Crossref PubMed Google Scholar). Second, Hcy-thiolactone is a reactive intermediate that causes protein N-homocysteinylation through the formation of amide bonds with ϵ-amino groups of protein lysine residues (5Jakubowski H. J. Biol. Chem. 1997; 272: 1935-1941Abstract Full Text Full Text PDF PubMed Google Scholar, 7Jakubowski H. Zhang L. Bardeguez A. Aviv A. Circ. Res. 2000; 87: 45-51Crossref PubMed Scopus (263) Google Scholar, 21Jakubowski H. FASEB J. 1999; 13: 2277-2283Crossref PubMed Scopus (325) Google Scholar, 22Jakubowski H. J. Biol. Chem. 2002; 277: 30425-30428Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Resulting protein damage necessitates the removal of N-homocysteinylated proteins by proteolytic degradation, which would further deplete cellular energy and limit cell growth. Hcy-thiolactone appears to be more toxic to human cells than Hcy (17Roybal C.N. Yang S. Sun C.W. Hurtado D. van der Jagt D.L. Townes T.M. Abcouwer S.F. J. Biol. Chem. 2004; 279: 14844-14852Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Hcy-containing proteins are also toxic (3Jakubowski H. J. Nutr. 2006; 136: 1741S-1749SCrossref PubMed Google Scholar, 24Jakubowski H. Clin. Chem. Lab. Med. 2005; 43: 1011-1014Crossref PubMed Scopus (63) Google Scholar) and induce an autoimmune response, which is associated with atherosclerosis in humans (1Jakubowski H. Cell. Mol. Life Sci. 2004; 61: 470-487Crossref PubMed Scopus (210) Google Scholar, 3Jakubowski H. J. Nutr. 2006; 136: 1741S-1749SCrossref PubMed Google Scholar, 24Jakubowski H. Clin. Chem. Lab. Med. 2005; 43: 1011-1014Crossref PubMed Scopus (63) Google Scholar, 25Undas A. Perła J. £aciński M. Trzeciak W. Kaźmierski R. Jakubowski H. Stroke. 2004; 35: 1299-1304Crossref PubMed Scopus (121) Google Scholar, 26Undas A. Jankowski M. Padjas A. Jakubowski H. Szczeklik A. Thromb. Haemost. 2005; 93: 346-350Crossref PubMed Scopus (57) Google Scholar).To minimize Hcy-thiolactone toxicity, cells had to evolve the mechanism of its disposal. Indeed, in all organisms, the bulk of Hcy-thiolactone is eliminated by excretion from cells into the extracellular media. In mice and humans, Hcy-thiolactone (27Chwatko G. Jakubowski H. Anal. Biochem. 2005; 337: 271-277Crossref PubMed Scopus (97) Google Scholar) is cleared out from the circulation by urinary excretion in the kidney (3Jakubowski H. J. Nutr. 2006; 136: 1741S-1749SCrossref PubMed Google Scholar, 28Chwatko G. Jakubowski H. Clin. Chem. 2005; 52: 408-415Crossref Scopus (69) Google Scholar). Hcy-thiolactone can also be disposed of by enzymatic hydrolysis by the serum Hcy-thiolactonase/paraoxonase (PON1) carried on high density lipoprotein (29Jakubowski H. J. Biol. Chem. 2000; 275: 3957-3962Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 30Jakubowski H. Ambrosius W. Pratt J.H. FEBS Lett. 2001; 491: 35-39Crossref PubMed Scopus (96) Google Scholar, 31£acinski M. Skorupski W. Sokolowska J. Cieslinski A. Trzeciak W.H. Jakubowski H. Cell. Mol. Biol. 2004; 50: 885-893PubMed Google Scholar, 32Domagała T.B. £acinski M. Trzeciak W.H. Mackness B. Mackness M.I. Jakubowski H. Cell. Mol. Biol. 2006; 52 (in press)PubMed Google Scholar).Since the serum PON1 is present extracellularly, it was unknown whether Hcy-thiolactone can be detoxified intracellularly. The present work describes intracellular Hcy-thiolactonase (HTLase) in humans and yeast and provides evidence that the HTLase is identical with bleomycin hydrolase (BLH), whose natural substrate and function were unknown. Purified human or yeast HTLase/BLH exhibits catalytic efficiency of 103 m−1 s−1 in the hydrolysis of Hcy-thiolactone, ∼100-fold greater than the catalytic efficiency of human serum Hcy-thiolactonase. Our data suggest that BLH is a major intracellular Hcy-thiolactone-hydrolyzing enzyme that protects cells against Hcy toxicity.EXPERIMENTAL PROCEDURESStrains and Plasmids—Saccharomyces cerevisiae strains used are listed in Table 1. Plasmids encoding hBLH and yBLH, and their enzymatically inactive variants C73A and H369A, respectively, were kindly provided by Leemor Joshua-Tor and Paul O'Farrell (35O'Farrell P.A. Gonzalez F. Zheng W. Johnston S.A. Joshua-Tor L. Struct. Fold. Des. 1999; 7: 619-627Abstract Full Text Full Text PDF Scopus (58) Google Scholar, 36Zheng W. Johnston S.A. Joshua-Tor L. Cell. 1998; 93: 103-109Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Escherichia coli BL21 (DE3) was used as a host for plasmid maintenance and recovery.TABLE 1S. cerevisiae strains used in this studyStrainGenotypeSourceHWY22MATa his3-1 leu2-0 met15-0 ura3-0/pYES2D. RamotarHWY23MATa his3-1 leu2-0 met15-0 ura3-0/pYES2-BLH1D. RamotarHWY24MATa his3-1 leu2-0 met15-0 ura3-0 blh1Δ::KanMX/pYES2D. RamotarW303-1AΔBLH1MATa ade2-1 can1-100 his3-11 leu2-3,112 trp1-1 ura3-1 blh1Δ::URA3D. RamotarXJB3-1BMATα met6 gal2Yeast Genetic Stock CenterABJ6-9MATα met6 leu2-3,112 trp1-1 blh1Δ::URA3This laboratoryABJ6-28MATα met6 leu2-3,112 trp1-1This laboratoryYS18MATα ura3-1 his3-11,15 leu2-3,112 CANrD. H. WolfYS18-ΔBLH1MATα ura3-1 his3-11,15 leu2-3,112 CANr blh1 Δ::KanMXD. Ramotar Open table in a new tab [35S]Hcy-thiolactone—Carrier-free l-[35S]Met (Amersham Biosciences) was supplemented with unlabeled l-methionine (Sigma) to a specific activity of 40,000 Ci/mol, converted to [35S]Hcy-thiolactone by a 4-h digestion with hydriodic acid at 128 °C, and purified by two-dimensional TLC as previously described (7Jakubowski H. Zhang L. Bardeguez A. Aviv A. Circ. Res. 2000; 87: 45-51Crossref PubMed Scopus (263) Google Scholar, 23Glowacki R. Jakubowski H. J. Biol. Chem. 2004; 279: 10864-10871Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 29Jakubowski H. J. Biol. Chem. 2000; 275: 3957-3962Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar).Purification of Native Yeast Hcy-thiolactone Hydrolase (yHTLase)—All steps were carried out at 4 °C. Fresh yeast cake (1 kg) was taken up in 10 mm potassium phosphate buffer (pH 6.8), 0.5 mm mercaptoethanol, 5% glycerol (3 liters) and disrupted in a high pressure homogenizer. The crude extract was clarified by centrifugation, mixed with DEAE-Sephacel (0.8 liters), and poured into a 5 × 40-cm column. The column was washed with the pH 6.8 buffer (4 liters) followed by a linear gradient of 0–0.5 m KCl in the pH 6.8 buffer (8 liters). At pH 6.8, HTLase activity is not retained on DEAE-Sephacel and elutes in the breakthrough fractions. Protein precipitated from these fractions with 60% ammonium sulfate was collected by centrifugation, dissolved in 50 mm potassium phosphate buffer (pH 6.8), and purified by Superdex 200 gel filtration. Active fractions were dialyzed against 50 mm Tris/HCl (pH 8.7), 0.5 mm mercaptoethanol, 5% glycerol and applied on a DEAE-Sephacel column. The yHTLase was eluted with a linear 0–0.5 m KCl gradient in the pH 8.7 buffer (Fig. 1, A and B). Pure yHTLase migrates on SDS-polyacrylamide gels as a 48-kDa protein (Fig. 1D).Purification of Native Human Placenta Hcy-thiolactone Hydrolase—All steps were carried out at 4 °C. Human placenta (100 g) was homogenized in 20 mm potassium phosphate buffer (pH 6.8), 0.5 mm mercaptoethanol, 5% glycerol (0.2 liters). The homogenate was clarified by centrifugation, and a protein fraction precipitated between 50 and 70% ammonium sulfate saturation was collected. Hcy-thiolactone-hydrolyzing activity was further purified by ion exchange chromatography on DEAE-Sephacel, gel filtration on Superdex 200, and chromatography on a hydroxyapatite column. Purification to homogeneity was achieved by preparative electrophoresis on nondenaturing polyacrylamide gels. Purified hHTLase migrated on SDS-polyacrylamide gels as a 48-kDa protein (Fig. 1D).MALDI-TOF Mass Spectrometric Analysis of Tryptic Peptides—Samples of native hBLH and yBLH were digested overnight at 37 °C with sequencing grade trypsin (Sigma) in 0.1 m ammonium bicarbonate. Peptide mass analysis was performed at the Autoflex mass spectrophotometer (Bruckner Daltonics, Leipzig, Germany) at the proteomics facility of the Institute of Biochemistry and Biophysics, Warsaw, Poland). Proteins were identified by the use of the MASCOT Server 1.9 based on mass searches within human and yeast sequences.Purification of Recombinant Human and Yeast Bleomycin Hydrolases—Plasmid encoding His-tagged hBLH or yBLH (35O'Farrell P.A. Gonzalez F. Zheng W. Johnston S.A. Joshua-Tor L. Struct. Fold. Des. 1999; 7: 619-627Abstract Full Text Full Text PDF Scopus (58) Google Scholar, 36Zheng W. Johnston S.A. Joshua-Tor L. Cell. 1998; 93: 103-109Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) was transformed into E. coli strain BL21 (DE3) for protein expression. The cells were grown in LB medium (0.6 liters) to midlog phase at 37 °C, the culture was shifted to 25 °C, and the BLH expression was induced with 0.5 mm isopropyl-β-d-thiogalactopyranoside for 16 h. The cells were harvested; resuspended in 50 mm potassium phosphate, pH 8.5, 300 mm NaCl, 7 mm 2-mercaptoethanol; and frozen at −80 °C. For BLH purification, the cells were thawed and disrupted by sonication on ice. Crude extracts were clarified by centrifugation at 4 °C, and BLH was purified by affinity chromatography on a 1-ml Ni2+-agarose (Amersham Biosciences) column. Pure BLH, eluted with 0.2 m imidazole, was dialyzed against 50 mm potassium phosphate buffer, pH 7.4, 7 mm 2-mercaptoethanol, 10% glycerol and stored at −20 °C.Preparation of Yeast Cell Extracts—Yeast cells from 10-ml cultures at 107 cells/ml were collected by centrifugation at 2 °C and disrupted by vortexing with glass beads (100–400 μm; Sigma) in 50 μl of ice-cold buffer (50 mm potassium phosphate, pH 7.5, 1 mm EDTA, 1 mm dithiothreitol; 3 × 0.5 min with 1-min cooling on ice intervals). Crude cells extracts were clarified by centrifugation using a JA25.50 rotor in a Beckman J2 centrifuge (30,000 × g, 15 min, 2 °C) and assayed for Hcy-thiolactonase activity.Enzyme Assays—Unless indicated otherwise, incubations were carried out at 25 °C in 50 mm potassium phosphate buffer (pH 7.4), 1 mm dithiothreitol, 1 mm EDTA. Hcy-thiolactonase activity was determined by following the formation of [35S]Hcy from [35S]Hcy-thiolactone as previously described (29Jakubowski H. J. Biol. Chem. 2000; 275: 3957-3962Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Hcy-thiolactonase activity was also assayed with unlabeled l-Hcy-thiolactone or its analogue by monitoring changes in UV absorption at A240 using a Varian Cary 50 UV-visible spectrophotometer (29Jakubowski H. J. Biol. Chem. 2000; 275: 3957-3962Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar).During enzyme purification, the HTLase activity was monitored by a TLC-based assay. Reaction mixtures (25 μl) contained 50 mm potassium phosphate (pH 7.5), 10 mm l-Hcy-thiolactone, 1 mm dithiothreitol, and a protein fraction (5 μl). Incubation was carried out at 37 °C. At the appropriate time, the reaction was stopped by transferring 3-μl aliquots onto the origin of a TLC plate (aluminum precoated with silica gel containing fluorescent indicator; Merck). The plate was developed with ethyl acetate/isopropyl alcohol/ammonia/water (27:23:5:3 by volume) for 15–20 min, dried, and visualized under UV light. In assays containing HTLase activity, dark spots of Hcy-thiolactone (Rf = 0.4) diminished or disappeared completely (Fig. 1B).Aminopeptidase activity was assayed with 0.1 mm arginine aminomethylcoumarylamide (R-AMC). After TLC separation, AMC, a highly fluorescent product of R-AMC hydrolysis, was visualized under UV light (Fig. 1C).In experiments in which utilization of other compounds (10 mm) was tested, potential substrates and products were separated by TLC and visualized by staining with ninhydrin or under UV. With all potential substrate-product pairs, complete separation was achieved on cellulose plates (Analtech) using 1-butanol/acetic acid/water (4:1:1 by volume) as a solvent (5Jakubowski H. J. Biol. Chem. 1997; 272: 1935-1941Abstract Full Text Full Text PDF PubMed Google Scholar, 29Jakubowski H. J. Biol. Chem. 2000; 275: 3957-3962Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar).Determination of Hcy-thiolactone in Yeast Cultures—Yeast cultures were maintained on minimal media for 24 h at 30 °C. Aliquots of the cultures (50 μl) were clarified by microcentrifugation (14,000 × g, 2 min, 25 °C). Cell-free media (2 μl) were diluted 20-fold with water, and half of the sample was applied onto a cation exchange HPLC column.HPLC Analyses—HPLC was carried out using a cation exchange polysulfoethyl aspartamide column (1.0 × 150 mm, 5 μm, 300 Å) from PolyLC, Inc. and System Gold Nouveau HPLC instrumentation from Beckman as previously described (27Chwatko G. Jakubowski H. Anal. Biochem. 2005; 337: 271-277Crossref PubMed Scopus (97) Google Scholar, 28Chwatko G. Jakubowski H. Clin. Chem. 2005; 52: 408-415Crossref Scopus (69) Google Scholar). After sample (20 μl) application the column was eluted isocratically with 10 mm sodium phosphate buffer, pH 6.6, 25 mm NaCl at a flow rate 0.15 ml/min. A postcolumn derivatization and fluorescence detection was used for the quantification of Hcy-thiolactone (eluting at 8 min) as previously described (27Chwatko G. Jakubowski H. Anal. Biochem. 2005; 337: 271-277Crossref PubMed Scopus (97) Google Scholar, 28Chwatko G. Jakubowski H. Clin. Chem. 2005; 52: 408-415Crossref Scopus (69) Google Scholar). The HPLC column effluent was mixed in a three-way tee with 2.5 mm o-phthaldiladehyde, 0.25 m NaOH, delivered at a flow rate of 0.07 ml/min. The mixture was passed through Teflon tubing reaction coil (0.3 mm I.D. x 3 m) and then was monitored with a Jasco 1520 fluorescence detector using excitation at 370 nm and fluorescence emission at 480 nm.RESULTSHuman and Yeast Hcy-thiolactonases Are Identical with Corresponding Bleomycin Hydrolases—In order to answer a question whether Hcy-thiolactone can be detoxified inside cells, we carried out a systematic search for an intracellular HTLase. We have examined HTLase activity levels in several tissues and found that human placenta is a better source of this activity than porcine liver. We have purified Hcy-thiolactone-hydrolyzing activity to homogeneity from human placenta. We have also purified to homogeneity an Hcy-thiolactone-hydrolyzing enzyme from the yeast S. cerevisiae (Fig. 1).To determine their identity, the purified human and yeast HTLases were digested with trypsin, and the resulting peptides were subjected to MALDI-TOF mass spectrometric analysis. The identified peptides from each enzyme were then used to search the NCBI data base. Mascot Server 1.9 (Bruckner Daltonics) searches revealed that the best match for human HTLase was human bleomycin hydrolase (hBLH, accession number gi|1321858). Sequence coverage was 72%, and the score was 1659. The score is the negative logarithm of the probability that the observed match is a random set. A similar analysis of the yeast HTLase revealed that the best match for the yeast enzyme was yeast bleomycin hydrolase (yBLH, accession number gi|3891714) encoded by the BLH1 gene, also known as GAL6 or LAP3. Sequence coverage was 54%, and the score was 2536.To confirm the identity of the human and yeast HTLases, we examined recombinant wild type and mutant clones, C73S of hBLH and H369A of yBLH (35O'Farrell P.A. Gonzalez F. Zheng W. Johnston S.A. Joshua-Tor L. Struct. Fold. Des. 1999; 7: 619-627Abstract Full Text Full Text PDF Scopus (58) Google Scholar, 36Zheng W. Johnston S.A. Joshua-Tor L. Cell. 1998; 93: 103-109Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), for the ability to hydrolyze Hcy-thiolactone. Plasmids encoding BLH proteins under the control of the β-galctosidase promoter were transformed into E. coli strain BL21(pLysS) for protein expression. Cells were grown in LB medium to midlog phase and induced with isopropyl-β-thiogalactosyl pyranoside. To assay HTLase activity, 10 mm Hcy-thiolactone was added to 0.1-ml aliquots of the cultures. Only cells expressing active variants of yBLH or hBLH completely hydrolyzed Hcy-thiolactone in 2 h at 30°C. No hydrolysis of Hcy-thiolactone was observed by uninduced cells (in the absence of isopropyl-β-d-thiogalactopyranoside) or induced cells expressing inactive H369A or C73S BLH variants. We have confirmed by SDS-PAGE of bacterial cell extracts that both active and inactive BLH variants were expressed at similar levels after isopropyl-β-d-thiogalactopyranoside induction. E. coli cells alone do not possess any significant ability to hydrolyze Hcy-thiolactone.Next, we purified recombinant hBLH and yBLH from the overproducing E. coli strains and found that the recombinant BLH proteins have the ability to hydrolyze HTL in vitro. We then reassayed DEAE-Sephacel fractions from our native HTLase purification procedures using R-AMC, an artificial substrate for BLH. We found that the yBLH aminopeptidase activity (Fig. 1C) correlated with yHTLase activity (Fig. 1B). Similar correlation between HTLase and BLH aminopeptidase activities was observed during the purification of hHTLase (not shown). We also found that BLH inhibitors, such as trans-epoxysuccinyl-l-leucilamido-(4-guanidino)butane (E-64), iodoacetate, or zinc (37Bromme D. Rossi A.B. Smeekens S.P. Anderson D.C. Payan D.G. Biochemistry. 1996; 35: 6706-6714Crossref PubMed Scopus (95) Google Scholar) also inhibited HTLase activities of hBLH and yBLH (Table 2). Dipeptides were also found to be inhibitors of the yHTLase activity (Table 2). Taken together, these results exclude the possibility that the HTLase activity may be due to a contaminating enzyme and show that the ability to hydrolyze HTL is an intrinsic property of the human as well as the yeast BLH.TABLE 2Inhibitors of the yeast and human HTLasesTested compoundInhibitionyHLTasehHTLase%%ZnCl2, 2 mm69.8NDCdCl2, 2 mm5.2NDCuCl2, 2 mm9.1NDIodoactamide, 2 mm94.099.9H2O2, 2 mm41.3NDE-64, 12.5 μm99.514.6E-64, 62.5 μmND94.4E-64, 125 μm98.797.1Arg-Ala, 10 mm86.0NDLys-Ala, 10 mm77.4NDLys-Leu, 10 mm26.4NDLeu-Ala, 10 mm50.0ND Open table in a new tab Substrate Specificity—Substrate specificity studies, summarized in Table 3, indicate that the hHTLase and yHTLase exhibit a high specificity for the l-stereoisomer of Hcy-thiolactone; d-Hcy-thiolactone was not hydrolyzed by any of the enzymes. For both enzymes, nonsaturating kinetics were observed for up to 20 mm l-Hcy-thiolactone, which precluded determinations of individual kcat and Km values. Catalytic efficiency values, kcat/Km, were obtained from the slopes of linear plots of initial l-Hcy-thiolactone hydrolysis rates divided by enzyme concentration versus l-Hcy-thiolactone concentrations. The hHTLase and yHTLase had similar catalytic efficiencies of 103 m−1 s−1 in the hydrolysis of l-Hcy-thiolactone, ∼100-fold greater than the catalytic efficiency of human serum Hcy-thiolactonase (29Jakubowski H. J. Biol. Chem. 2000; 275: 3957-3962Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). For comparison, the catalytic efficiencies of hBLH and yBLH for the degradation of bleomycin A2 are 5000 m−1 s−1 (37Bromme D. Rossi A.B. Smeekens S.P. Anderson D.C. Payan D.G. Biochemistry. 1996; 35: 6706-6714Crossref PubMed Scopus (95) Google Scholar) and 4.6 m−1 s−1 (calculated from the data of Ref. 38Pei Z. Calmels T.P. Creutz C.E. Sebti S.M. Mol. Pharmacol. 1995; 48: 676-681PubMed Google Scholar). l-Homoserine-lactone was not hydrolyzed. γ-Thiobutyrolactone and N-acetyl-dl-Hcy-thiolactone also were not hydrolyzed, which shows that the α-amino group is essential for hydrolysis of l-Hcy-thiolactone. The hHTLase and yHTLase also hydrolyzed methyl esters of l-Cys and l-Met; however, methyl esters of α-l-Ala, β-l-Ala, l-Lys, and l-Trp were not hydrolyzed (Table 3).TABLE 3Sub" @default.
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