Conserved Residues in the Putative Catalytic Triad of Human Bile Acid Coenzyme A:Amino Acid N-Acyltransferase

Human bile acid-CoA:amino acidN-acyltransferase (hBAT), an enzyme catalyzing the conjugation of bile acids with the amino acids glycine or taurine has significant sequence homology with dienelactone hydrolases and other α/β hydrolases. These enzymes have a conserved catalytic triad that maps onto the mammalian BATs at residues Cys-235, Asp-328, and His-362 of the human sequence, albeit that the hydrolases contain a serine instead of a cysteine. In the present study, the function of the putative catalytic triad of hBAT was examined by chemical modification with the cysteine alkylating reagent N-ethylmaleimide (NEM) and by site-directed mutagenesis of the triad residues followed by enzymology studies of mutant and wild-type hBATs. Treatment with NEM caused inactivation of wild-type hBAT. However, preincubation of wild-type hBAT with the substrate cholyl-CoA before NEM treatment prevented loss of N-acyltransferase activity. Substitution of His-362 or Asp-328 with alanine results in inactivation of hBAT. Although substitution of Cys-235 with serine generated an hBAT mutant with lower N-acyltransferase activity, it substantially increased the bile acid-CoA thioesterase activity compared with wild type. In summary, data from this study support the existence of an essential catalytic triad within hBAT consisting of Cys-235, His-362, and Asp-328 with Cys-235 serving as the probable nucleophile and thus the site of covalent attachment of the bile acid molecule. Human bile acid-CoA:amino acidN-acyltransferase (hBAT), an enzyme catalyzing the conjugation of bile acids with the amino acids glycine or taurine has significant sequence homology with dienelactone hydrolases and other α/β hydrolases. These enzymes have a conserved catalytic triad that maps onto the mammalian BATs at residues Cys-235, Asp-328, and His-362 of the human sequence, albeit that the hydrolases contain a serine instead of a cysteine. In the present study, the function of the putative catalytic triad of hBAT was examined by chemical modification with the cysteine alkylating reagent N-ethylmaleimide (NEM) and by site-directed mutagenesis of the triad residues followed by enzymology studies of mutant and wild-type hBATs. Treatment with NEM caused inactivation of wild-type hBAT. However, preincubation of wild-type hBAT with the substrate cholyl-CoA before NEM treatment prevented loss of N-acyltransferase activity. Substitution of His-362 or Asp-328 with alanine results in inactivation of hBAT. Although substitution of Cys-235 with serine generated an hBAT mutant with lower N-acyltransferase activity, it substantially increased the bile acid-CoA thioesterase activity compared with wild type. In summary, data from this study support the existence of an essential catalytic triad within hBAT consisting of Cys-235, His-362, and Asp-328 with Cys-235 serving as the probable nucleophile and thus the site of covalent attachment of the bile acid molecule. In humans, the majority of bile acids (BAs) 1The abbreviations used are: BA(s), bile acid(s); BAT, bile acid CoA:amino acid N-acyltransferase; DTT, dithiothreitol; ESI-MS, electrospray ionization-mass spectrometry; LC-ESI-MS-MRM, liquid chromatography-ESI-MS-multiple reaction monitoring; MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight; IPTG, isopropyl- 1-thio-β-d-galactopyranoside synthesized by the liver are conjugated with glycine or taurine, a reaction that favors their excretion into bile (1Zouboulis-Vafiadis I. Dumont M. Erlinger S. Am. J. Physiol. 1982; 243: G208-G213Google Scholar, 2Vessey D.A. Whitngy J. Gollan J.L. Biochem. J. 1983; 214: 923-927Google Scholar) and uptake from portal blood into the liver (3Schroeder A. Eckhardt U. Stieger B. Tynes R. Schteingart C.D. Hofmann A.F. Meier P.J. Hagenbuch B. Am. J. Physiol. 1998; 274: G370-G375Google Scholar). Conjugation also promotes absorption of fat and fat-soluble vitamins A, D, E, and K in the acidic environment of the small intestine by lowering the pK a of bile acids and hence maintaining BA solubility. Conjugation of bile acids with amino acids occurs in two steps. In the first, BAs form a thioester with CoA, catalyzed by BA-CoA ligase. In the second step, or amidation reaction, either glycine or taurine is conjugated to BA-CoA to form a BA amidate and CoA is displaced. The amidation reaction (ReactionFR1) is catalyzed by bile acid CoA:amino acid N-acyltransferase (BAT). BAT has been purified from the livers of rats (4Killenberg P.G. Jordan J.T. J. Biol. Chem. 1978; 253: 1005-1010Google Scholar), cows (5Vessey D.A. J. Biol. Chem. 1979; 254: 2059-2063Google Scholar), chickens (6Czuba B. Vessey D.A. Biochem. J. 1981; 195: 263-266Google Scholar), and humans (7Kimura M. Okuno E. Inada J. Ohyama H. Kido R. Hoppe-Seyler's Z. Physiol. Chem. 1983; 364: 637-645Google Scholar, 8Johnson M. Barnes S. Kwakye J. Diasio R.B. J. Biol. Chem. 1991; 266: 10227-10233Google Scholar). The human (9Falany C.N. Johnson M.R. Barnes S. Diasio R.B. J. Biol. Chem. 1994; 269: 19375-19379Google Scholar) and mouse BAT (10Falany C.N. Fortinberry H. Leiter E.H. Barnes S. J. Lipid Res. 1997; 38: 1139-1148Google Scholar) genes have been cloned, and the enzymatically active recombinant enzymes expressed in bacteria. A putative rat BAT (Kan-1) has been described (11Furutani M. Arii S. Higashitsuji H. Mise M. Fukumoto M. Takano S. Nakayama H. Imamura M. Fujita J. Biochem. J. 1995; 311: 203-208Google Scholar); however, the expression and enzymatic characterization of Kan-1 have not been reported. Sequence similarity searches at the NCBI BLAST server (www.ncbi.nlm.nih.gov/BLAST/) against non-redundant protein data base using the iterative algorithm PSI-BLAST (12Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Google Scholar) have detected significant similarity of BAT to dienelactone hydrolase and other α/β hydrolases of known structure. The α/β hydrolase fold is shared by several enzymes that apparently have diverged from a common ancestor. Despite their different catalytic functions, these enzymes all contain a conserved nucleophile-histidine-acid catalytic triad with the histidine being a completely conserved amino acid and the nucleophile and acid loops accommodating more than one type of amino acid (13Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington S.J. Silman I. Schrag J. Sussman J.L. Verschueren K.H.G. Goldman A. Protein Eng. 1992; 5: 197-221Google Scholar). Amino acid sequence alignments also revealed that hBAT and other BATs have high homology (above or equal to 40%) with peroxisomal, mitochondrial, and cytosolic long chain acyl-CoA thioesterases or acyl-CoA thioesterases. The conserved amino acid residues are indicated in Fig. 1. Using mutation analysis, Huhtinen et al. established the serine-histidine-aspartic acid triad in CTE-I (14Huhtinen K. O' Byrne J. Lindquist P.J.G. Contreras J.A. Alexson S.E.H. J. Biol. Chem. 2002; 277: 3424-3432Google Scholar). The importance of these residues had been suggested by previous site-directed mutagenesis experiments, and the enzyme reaction mechanism speculated (15Pazirandeh M. Chirala S.S. Wakil S.J. J. Biol. Chem. 1991; 266: 20946-20952Google Scholar, 16Witkowski A. Naggert J. Wessa B. Smith S. J. Biol. Chem. 1991; 266: 18514-18519Google Scholar, 17Witkowski A. Naggert J. Witkowska H.E. Randhawa Z.I. Smith S. J. Biol. Chem. 1992; 267: 18488-18492Google Scholar). Furthermore, when the active site-serine was substituted with a cysteine the thioesterase II, a fatty acyl-thioester hydrolase, was converted to an acyltransferase (18Witkowski A. Witkowska H.E. Smith S. J. Biol. Chem. 1994; 269: 379-383Google Scholar). Thus, we have hypothesized that hBAT utilizes a catalytic triad composed of Cys-235, His-362, and Asp-328 with Cys-235 as the nucleophile. In this report we describe experiments to support the hypothesis that Cys-235, His-362, and Asp-328 are essential for theN-acyltransferase activity of hBAT and form the catalytic triad with Cys-235 serving as the nucleophile. Replacement of Cys-235 by serine converted hBAT from an N-acyltransferase into a bile acid-CoA thioesterase, which is consistent with a reaction mechanism based on the formation of a bile acid-hBAT covalent intermediate via a thioester bond. These results also demonstrate the close relationship between acyl-CoA thioesterases andN-acyltransferases. Cholic acid was purchased from Sigma. NEM was from Research Organics (Cleveland, OH). The QuikChange site-directed mutagenesis kit was obtained from Stratagene (La Jolla, CA). Oligonucleotides were synthesized by Sigma-Genosys (The Woodlands, Texas). [2-3H]Taurine (29 Ci/mmol) and [2-3H]glycine (10 Ci/mmol) were purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO) and Amersham Biosciences, respectively. QIAquick Gel Extraction Kit and QIAprep Spin Miniprep Kit were purchased from Qiagen (Valencia, CA). Centricon Centrifugal Filter Devices were obtained from Millipore (Bedford, MA). Restriction enzymes were purchased from New England Biolabs (Beverly, MA) and Promega (Madison, WI). Sep-Pak Plus C18 cartridges were purchased from Waters (Milford, MA). pET-21a(+) and Bugbuster protein extraction reagent were obtained from Novagen (Madison, WI). The Rapid Ligation Kit was from Roche Molecular Biochemicals. SoftLink Avidin Resin was purchased from Promega. Bacterial cytosol containing wild-type hBAT (150 μg of total protein) was incubated with NEM (680 μm) for 2, 5, and 10 min at 37 °C. At the end of the incubations, dithiothreitol (DTT; 680 μm) was added to inactivate residual NEM.N-acyltransferase activity was then determined following addition of cholyl-CoA (0.5 mm), [2-3H]taurine (0.1 mm) and phosphate buffer (100 mm K2HPO4, pH 8.25) and incubation at 37 °C for 30 min. The total reaction volume was 100 μl. Different concentrations of NEM (50, 100, 200, 400, 600, and 800 μm) were incubated with bacterial cytosol containing wild-type hBAT (150 μg of total protein) for 10 min at 37 °C.N-Acyltransferase activity was then determined as previously described. Bacterial cytosol containing wild-type hBAT (150 μg of total protein) was preincubated with cholyl-CoA (1 mm) in phosphate buffer for 0, 2, and 5 min at 37 °C, and then NEM (680 μm) was added. The mixture was incubated for a further 10 min at 37 °C followed by the addition of DTT (680 μm).N-Acyltransferase activity was determined with the addition of [2-3H]taurine (0.05 μCi, 0.1 mm). The total reaction volume was 120.5 μl. Cholyl-CoA was chemically synthesized as described previously (19Johnson M. Barnes S. Diasio R.B. Anal. Biochem. 1989; 182: 360-365Google Scholar), using a modification of the method of Shah and Staple (20Shah P.P. Staple E. Steroids. 1968; 12: 571-576Google Scholar). Cholyl-CoA was first separated from the reaction mixture by three extractions with five reaction volumes of ether in a separating funnel. The aqueous phase was collected and purified by absorption onto Sep-Pak Plus C18 cartridges. After the sample was loaded, the cartridge was washed with 3 × 3 ml of 20% methanol in double distilled H2O. Elution was carried out with 10 mm NH4HCO3 in methanol. After removal of the elution buffer by evaporation into air, cholyl-CoA was dissolved in double-distilled H2O, and its concentration determined spectrophotometrically. An ε259 nm = 16,800m−1 cm−1 was used to calculate the concentration. Since it was difficult to detect low levels of cholate contamination in cholyl-CoA by high pressure liquid chromatography-based procedures, electrospray ionization-mass spectrometry (ESI-MS) was used to confirm the purity of the synthesized cholyl-CoA. Analysis was carried out on a PE-Sciex (Concord, Ontario, Canada) API III triple quadrupole mass spectrometer. The sample was introduced into the mass spectrometer via the electrospray ionization interface with an ionizing needle voltage of –4900 V and an orifice potential of –60 V. Negative ion spectra were recorded over a range ofm/z values from 190 to 2000. The purity of the cholyl-CoA preparation by this analysis was estimated to be better than 99.5%. The full-length cDNA for hBAT was cloned into the pKK233–2 vector as described previously (9Falany C.N. Johnson M.R. Barnes S. Diasio R.B. J. Biol. Chem. 1994; 269: 19375-19379Google Scholar). Plasmid pET-21a(+)/hBAT was constructed by first amplifying hBAT coding sequence by PCR with pKK233–2/hBAT as template DNA. Restriction enzyme sites NdeI and XhoI were generated at the 5′ and 3′ end, respectively. The PCR products were then digested with restriction enzymes NdeI and XhoI. The resulting fragment, containing the hBAT coding sequence, was then ligated into the similarly digested pETGagbiotinHis. The plasmid pETGagbiotinHis is a modified version of pET-21a(+) containing the coding sequence for a viral gag protein and a biotinylation tag. 2M. Sakalian, unpublished results. This peptide tag serves as the substrate for site-specific biotinylation by the biotin E. P.J. Protein Scholar). with NdeI and XhoI the gag sequence the biotinylation tag coding sequence to be to the 3′ end of the hBAT coding A QuikChange site-directed mutagenesis kit was used for all the in this The expression plasmid pKK233–2/hBAT or pET-21a(+)/hBAT was used as the template for Oligonucleotides used for the mutagenesis are in with the the template was by and the PCR products were and by sequence analysis, and plasmid the used for site-directed mutagenesis to the and hBAT are in a The are were into E. or the expression vector The were in ml of containing (100 or for expression of enzyme (100 and (10 in a at 37 °C with was added to a concentration of mm or mm with biotin in when the had to of The incubation was to 30 °C, and were by at × for min at °C. The were in Bugbuster protein extraction reagent using 5 ml of reagent of and of ml of the The was incubated on a at a for min at The was by at × for min at °C. The was for N-acyltransferase activity and at °C. was with an composed of phosphate mm), and mm) and then onto a ml with Bugbuster protein extraction reagent The was used to hBAT from the bacterial to hBAT was a for hBAT not The was washed with mm The protein was with 10 mm biotin in mm The was to for min before were Protein concentration was determined by the protein using as the Anal. Biochem. Scholar). activity the formation of bile acid (19Johnson M. Barnes S. Diasio R.B. Anal. Biochem. 1989; 182: 360-365Google Scholar). The reaction mixture cholyl-CoA mm), [2-3H]taurine or [2-3H]glycine (0.1 μCi, mm), and (100 mm), pH The reaction was by the addition of enzyme preparation to the reaction mixture and for 30 min at 37 °C. The reaction was by the addition of (100 mm) pH containing and (19Johnson M. Barnes S. Diasio R.B. Anal. Biochem. 1989; 182: 360-365Google Scholar). The reaction products were separated from taurine or glycine by extraction into the of the phase were with and the determined in a liquid The was based on the method described previously (14Huhtinen K. O' Byrne J. Lindquist P.J.G. Contreras J.A. Alexson S.E.H. J. Biol. Chem. 2002; 277: 3424-3432Google Scholar). The buffer mm 10 mm and mm cholyl-CoA and glycine were with the buffer to enzyme The total reaction volume was activity was by the at Cholyl-CoA μm) and glycine or taurine mm) were incubated with wild-type or mutant hBAT (1 in (100 mm), pH at 37 °C for 5 min. The total reaction volume was 100 μl. The reaction products were with 100 of by for min and by An phase × 100 mm) was A was 10 mm and buffer was 10 mm in The was from the with a of buffer and introduced into the electrospray ionization interface in the ion The ion for for for were to detect the formation of and A was generated with containing known concentrations of and The estimated of reaction and were determined by to the The K for bile acid-CoA thioesterase activity for wild-type and mutant hBAT were estimated using analysis of a double The results are as the of values and V values were carried out using a The in of hBAT were to peptide and of the were to and then in 10 of aqueous An (1 was with of a of acid in aqueous The mixture (1 was onto a and to mass spectrometry was on an mass spectrometer. The resulting mass spectrometry data was and significant for analysis using the at cysteine residues are to hBAT enzyme activity, bacterial cytosol containing wild-type hBAT was incubated with a cysteine for 2, 5, and 10 min at 37 °C. DTT was then added to with the residual were confirm the hBAT inactivation by NEM modification in different in a different concentrations of NEM were incubated with bacterial cytosol containing wild-type were similarly A that NEM incubation with wild-type hBAT caused a activity. a 10 min more than of activity was hBAT NEM incubation was used as a and had loss of activity. Fig. the of NEM concentrations activity. At more than of the activity was Since NEM modification caused loss of N-acyltransferase activity, one or more of the cysteine residues in hBAT be for enzyme activity and one be the active The be the loss of hBAT activity by with this cysteine Cholyl-CoA was preincubated with cytosol containing hBAT for 0, 2, and 5 min at 37 °C. NEM was added at the end of the and the mixture was incubated for an 10 min. DTT was then added to with the residual activity was of cytosol containing hBAT with its bile acid-CoA before the addition of NEM of BAT activity a first to the hypothesis that hBAT has a catalytic His-362 and Asp-328 were to In Cys-235 was to a serine serine is the nucleophile for the enzymes in the and which for Cys-235, His-362, and Asp-328, were to the and which for and respectively. These hBAT with wild-type were expressed in bacteria. levels were in A the of the mutant and wild-type hBAT. In the protein were as hBAT and biotin by mass spectrometry analysis of their hBAT was purified with the to based on The catalytic of these mutant and wild-type enzymes were using cholyl-CoA and taurine or glycine as the activity was detected from either the or However, the mutant bile acid-CoA thioesterase activity to the not In all of the substrate was based on the from = cm−1 for (14Huhtinen K. O' Byrne J. Lindquist P.J.G. Contreras J.A. Alexson S.E.H. J. Biol. Chem. 2002; 277: 3424-3432Google Scholar). Since inactivate wild-type hBAT by with its cysteine residues not analysis was used for of the mutant and wild-type hBAT for N-acyltransferase and thioesterase analysis revealed that the of hBAT has lower N-acyltransferase activity compared with wild however, an in the of of when glycine was used as the second was of thioesterase activity The thioesterase activity of hBAT was then in was and was wild-type hBAT had a K of and V of These were significant In the present study we have obtained to support the protein and the hypothesis that hBAT utilizes a catalytic triad composed of residues Cys-235, His-362, and Asp-328 with Cys-235 as the nucleophile. hBAT this catalytic the catalytic mechanism of hBAT the mechanism shared by serine J. Biochem. Scholar) and thioesterases by et al. (15Pazirandeh M. Chirala S.S. Wakil S.J. J. Biol. Chem. 1991; 266: 20946-20952Google Scholar). The from the ion of Asp-328 is to His-362 and then to Cys-235 to the of the nucleophile. In this a intermediate one intermediate 3 are for the catalytic Cys-235 as the was obtained using several The enzyme be by a that one or more cysteine residues are for the enzyme activity. Cholyl-CoA preincubation before NEM addition prevented of a that of the cysteine by an of substrate the enzyme from NEM mutagenesis of His-362 and Asp-328 to alanine in inactivation of hBAT that these two residues are for the enzyme activity. These results are also consistent with the of these residues as by sequence with enzymes and the hBAT protein as by to dienelactone hydrolase and other α/β hydrolases of known His-362 and Asp-328 are the histidine and residues conserved and dienelactone hydrolases studies have that these residues are for enzyme activity in thioesterases and α/β hydrolases (13Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington S.J. Silman I. Schrag J. Sussman J.L. Verschueren K.H.G. Goldman A. Protein Eng. 1992; 5: 197-221Google Scholar, K. O' Byrne J. Lindquist P.J.G. Contreras J.A. Alexson S.E.H. J. Biol. Chem. 2002; 277: 3424-3432Google Scholar, M. Chirala S.S. Wakil S.J. J. Biol. Chem. 1991; 266: 20946-20952Google Scholar, 16Witkowski A. Naggert J. Wessa B. Smith S. J. Biol. Chem. 1991; 266: 18514-18519Google Scholar, 17Witkowski A. Naggert J. Witkowska H.E. Randhawa Z.I. Smith S. J. Biol. Chem. 1992; 267: 18488-18492Google Scholar, A. Witkowska H.E. Smith S. J. Biol. Chem. 1994; 269: 379-383Google Scholar). Since the and residues are for the nucleophile and it to the cysteine their with alanine the the Cys-235 was to a the mutant enzyme had a lower N-acyltransferase activity. In its thioesterase activity was substantially is that an intermediate is the formation on the from either one of the second or when glycine was used for the serine from mutant the from is more than that from resulting in cholate as the and as a This also be used to the acid of taurine, is a substrate of hBAT (7Kimura M. Okuno E. Inada J. Ohyama H. Kido R. Hoppe-Seyler's Z. Physiol. Chem. 1983; 364: 637-645Google Scholar), and taurine are D.J. Barnes S. Diasio R.B. U. S. A. Scholar). The is also consistent with previous studies on in which the serine intermediate was to be to the cysteine these when the active site serine was to a cysteine and the mutant enzyme was incubated with the cysteine was (14Huhtinen K. O' Byrne J. Lindquist P.J.G. Contreras J.A. Alexson S.E.H. J. Biol. Chem. 2002; 277: 3424-3432Google Scholar, M. Chirala S.S. Wakil S.J. J. Biol. Chem. 1991; 266: 20946-20952Google Scholar). Although in hBAT is the other conserved site-directed mutagenesis suggested that being for BAT activity, it is to be the nucleophile in the enzyme was to an alanine the mutant hBAT significant N-acyltransferase activity. spectrometry analysis of the reaction products of mutant hBAT established that this mutant hBAT activity not amino acid sequence hBAT is to acyl-CoA thioesterases. The thioesterases a catalytic triad composed of with as the nucleophile. In a previous study with human has that hBAT bile acid-CoA thioesterase activity and be to long chain acyl-CoA thioesterases K. A. J. Lipid Res. Scholar, P.J. Alexson S.E.H. J. Biochem. 1998; Scholar, Alexson S.E.H. J. Biol. Chem. 1995; Scholar, M. Alexson S.E.H. J. Biochem. 1995; Scholar). an acyl-CoA thioesterase, when CoA of bile acids cholyl-CoA and used as its was K. Alexson S.E.H. J. Biol. Chem. 2002; 277: Scholar). The that for are on and the for are on mutant hBAT. al. have speculated the of hydrolase to via in the of a fatty acyl-thioester hydrolase, thioesterase (18Witkowski A. Witkowska H.E. Smith S. J. Biol. Chem. 1994; 269: 379-383Google Scholar). Although this is the first the mutation has been on the the close relationship between thioesterases and hydrolases is hBAT is one of the enzymes for bile acid which is for absorption of and fat as fat in human (3Schroeder A. Eckhardt U. Stieger B. Tynes R. Schteingart C.D. Hofmann A.F. Meier P.J. Hagenbuch B. Am. J. Physiol. 1998; 274: G370-G375Google Scholar). in the enzyme to of the of these essential for Protein for the protein of for hBAT from for the and and Johnson for with the

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