Báo cáo khoa học: Characterization of a novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis docx

Characterization of a novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis Puja Shahi* † , Ish Kumar* ‡ , Ritu Sharma, Shefali Sanger and Ravinder S. Jolly Institute of Microbial Technology, Chandigarh, India Long-chain acyl-CoA thioesterases (EC 3.1.2.2) hydro- lyze acyl-CoA esters to nonesterified fatty acids and coenzyme A (CoASH) [1]. These are ubiquitously expressed in bacteria, yeast, plants and mammals, and in most cell compartments, such as endoplasmic reticu- lum, cytosol, mitochondria and peroxisomes. Several unrelated thioesterases have been purified to homogen- eity from plants, animals, and bacteria, and the cDNAs encoding several of them have been cloned and sequenced [2–7]. Although the physiological func- tions of these enzymes remain largely unknown, it is speculated that they regulate lipid metabolism by maintaining appropriate concentrations of acyl-CoA, CoASH, and nonesterified fatty acids. The only estab- lished function for acyl-CoA thioesterases is in the termination of fatty acid synthesis in eukaryotes [8]. Two thioesterases, I and II, that cleave acyl-CoA molecules in vitro have been characterized from Keywords Alcaligenes faecalis; immunogold electron microscopy; long-chain acyl-CoA; p-nitrophenyl esters; thioesterase Correspondence R. S. Jolly, Institute of Microbial Technology, Sector 39, Chandigarh 160 036, India Fax: +91 172 269 0585 Tel: +91 172 269 0908 E-mail: jolly@imtech.res.in *These authors contributed equally to this paper †Present address Department of Physiology and Biophysics, University of Iowa, Iowa city, IA 52242, USA ‡Present address Department of Chemistry, Wesleyan Univer- sity, Middletown, CT 06459, USA (Received 19 January 2006, revised 12 March 2006, accepted 22 March 2006) doi:10.1111/j.1742-4658.2006.05244.x A novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis has been isolated and characterized. The protein was extracted from the cells with 1 m NaCl, which required 1.5-fold, single-step purification to yield near-homogeneous preparations. In solution, the protein exists as homo- meric aggregates, of mean diameter 21.6 nm, consisting of 22-kDa sub- units. MS ⁄ MS data for peptides obtained by trypsin digestion of the thiosterase did not match any peptide from Escherichia coli thioesterases or any other thioesterases in the database. The thioesterase was associated exclusively with the surface of cells as revealed by ultrastructural studies using electron microscopy and immunogold labeling. It hydrolyzed satur- ated and unsaturated fatty acyl-CoAs of C 12 to C 18 chain length with V max and K m of 3.58–9.73 lmolÆmin )1 Æ(mg protein) )1 and 2.66–4.11 lm, respect- ively. A catalytically important histidine residue is implicated in the active site of the enzyme. The thioesterase was active and stable over a wide range of temperature and pH. Maximum activity was observed at 65 °C and pH 10.5, and varied between 60% and 80% at temperatures of 25–70 °C and pH 6.5–10. The thioesterase also hydrolyzed p-nitrophenyl esters of C 2 to C 12 chain length, but substrate competition experiments demonstrated that the long-chain acyl-CoAs are better substrates for thio- esterase than p-nitrophenyl esters. When assayed at 37 and 20 °C, the affin- ity and catalytic efficiency of the thioesterase for palmitoleoyl-CoA and cis-vaccenoyl-CoA were reduced approximately twofold at the lower tem- perature, but remained largely unaltered for palmitoyl-CoA. Abbreviations DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid); TEM, transmission electron microscopy. 2374 FEBS Journal 273 (2006) 2374–2387 ª 2006 IMTECH Escherichia coli. Thioesterase I, encoded by the tesA gene, is a periplasmic protein of 20.5 kDa and has an active site similar to serine proteases, consistent with its inhibition with di-isopropyl fluorophosphate [2,9]. Thioesterase II, encoded by the tesB gene, is a tetra- meric protein with identical subunits of 32 kDa and is insensitive to inhibition with di-isopropyl fluorophos- phate. A histidine residue present at position 58 in thioest- erase II has been implicated in the cleavage of the thioester bond [10,11]. Two thioesterases with striking similarities in their physical properties to thioesterase I and II have been reported from photosynthetic bac- teria, Rhodopseudomonas sphaeroides [12,13]. Cho and Cronan [2] prepared null mutants of both tesA and tesB in an attempt to determine the role of these thioesterases. The deletion of tesA, tesB or both genes has no effect on the growth or lipid composition of E. coli. However, the possibility of another enzyme taking over the function of both enzymes or the pres- ence of a third thioesterase in E. coli has not been ruled out. The overexpression of either tesA or tesB to levels that greatly exceed normal also has no effect on the growth of E. coli [2,11]. It has also been shown that E. coli thioesterases are unable to cleave acyl- ACPs in vivo [14]. Recently, Schulz and coworkers [15] provided some evidence for the function of cytoplasmic thioesterase of E. coli in b-oxidation. They showed that oleate is mostly degraded via the classical, isomerase-dependent pathway in E. coli, but that a small amount of 2-trans- 5-cis-tetradecadienoyl-CoA is diverted from the path- way by conversion into 3,5-cis-tetradecadienoyl-CoA by D 3 ,D 2 -enoyl-CoA isomerase. The 3,5- intermediate, which would strongly inhibit b-oxidation if allowed to accumulate, is hydrolyzed and the resultant 3,5-tetra- decadienoate is excreted into the growth medium. In another study, Zheng et al. [16] coexpressed thioest- erase II with (R)-3-hydroxydecanoyl–acyl carrier pro- tein-CoA transacylase (PhaG, encoded by the phaG gene) to clarify the physiological role of thioesterase II. 3-Hydroxydecanoic acid was produced in E. coli by mobilizing PhaG. By using an isogenic tesB (encoding thioesterase II)-negative knockout E. coli strain, CH01, it was found that the expression of tesB and phaG can up-regulate each other. In addition, 3-hydroxydecanoic acid was synthesized from glucose or fructose by recombinant E. coli harboring phaG and tesB. This study supports the hypothesis that the physiological role of thioesterase II in E. coli is to prevent the abnormal accumulation of intracellular acyl-CoA. We have isolated a thioesterase from Alcaligenes faecalis ISH108 and demonstrated its application in chemoselective and racemization free deacylation of thiol esters [17]. A. faecalis was isolated from soil sam- ples during routine screening of micro-organisms for various biotransformation applications. In this paper, we describe the intracellular localization and character- ization of the thioesterase. The wild-type expression of protein was sufficiently large to obtain milligram quan- tities of the protein from about 20 g of cells, which required only 1.5-fold, single-step purification to obtain a near homogeneous preparation. Results Isolation and purification of thioesterase The thioesterase extracted from the cells with 1 m NaCl, as described in Experimental procedures, exhib- ited a specific activity of 4.72 lmolÆmin )1 Æ(mg pro- tein) )1 . The enzyme could also be extracted by suspending freshly grown cells in 50 mm Tris ⁄ HCl buffer saturated with butanol at pH 7.4 [specific activ- ity 2.5 lmolÆmin )1 Æ(mg protein) )1 ]. Other extraction systems, used to extract thioesterase from the cells, included 1 m NaCl with 0.1% Triton X-100 [specific activity 2.82 l molÆ min )1 Æ(mg protein) )1 ] and butan- 1-ol-saturated Tris ⁄ HCl buffer containing 0.1% Triton X-100 [specific activity 2.43 lmolÆmin )1 Æ(mg pro- tein) )1 ]. As the specific activity in 1 m NaCl extract was the highest, it was selected as the method of choice for the isolation of the enzyme. SDS ⁄ PAGE of the NaCl extract, run under reducing conditions, showed a prom- inent band (> 90%) at 22 kDa, which suggested that further purification of the protein could be achieved by size exclusion chromatography. Preliminary investiga- tion using Sephadex G-75 (fractionation range 3–80 kDa) revealed that the native size of the protein was much larger as it moved into the void volume of the column. This allowed ultrafiltration with a 50-kDa Centricon membrane (Amicon, Bedford, MA, USA) for concentration of the samples or buffer change. In the first attempt, Sephacryl S-300 (fractionation range 10–1500 kDa) was selected for the purification of protein. The NaCl extract obtained was desalted and concentrated using 50-kDa Centricon membranes. The concentrated sample was loaded on the column pre- equilibrated with 50 mm Tris ⁄ HCl buffer (pH 7.6) containing 150 mm NaCl. The column was eluted with the same buffer at a flow rate of 24 mLÆh )1 . Thioest- erase activity moved near the void volume of the col- umn (Fig. 1A). Finally, the protein was purified on a Sepharose CL-4B (fractionation range 60–20 000 kDa) column (Fig. 1B). SDS ⁄ PAGE of the purified protein, P. Shahi et al. Thioesterase of Alcaligenes faecalis FEBS Journal 273 (2006) 2374–2387 ª 2006 IMTECH 2375 run under reducing conditions, showed a single band at 22 kDa (Fig. 2A). Sepharose CL-4B chromatography resulted in 1.5-fold purification with  50% yield. Molecular mass of thioesterase The elution profile of the protein on gel filtration col- umns indicated the protein to be a large homomeric aggregate of 22-kDa subunits. On the Sepharose CL-4B column, the thioesterase was eluted just after the higher- molecular-mass (2000 kDa) fraction of dextran blue and much before the standard protein, thyroglobulin (669 kDa) (Fig. 1C). The inability of the protein to move in native PAGE (anodic disc-PAGE using Tris ⁄ glycine, as electrophoresis buffer, pH 8.3, or cathodic disc PAGE using alanine ⁄ acetic acid buffer, pH 4.5), run under non- reducing conditions, is also consistent with this observa- tion. Finally, the aggregated structure of native protein was established by transmission electron microscopy (TEM). The purified sample was concentrated by repeated ultrafiltration using a Centricon 50-kDa mem- brane and suspended in water at a concentration of 600 lgÆmL )1 . TEM was performed on a carbon grid using 2% aqueous uranyl acetate and 2% phosphotung- stic acid at pH 8.0. An electron micrograph showed granular structures with a mean diameter of 21.6 nm (Fig. 3A). The size distribution is shown in Fig. 3B. Intracellular localization of thioesterase We carried out electron microscopic immunogold labe- ling studies with ultrathin sections of Alcaligenes cells to localize thioesterase at the ultrastructural level. Polyclonal antibodies, AbTE-N and AbTE-D, raised against purified native enzyme and the piece of gel cor- responding to the 22-kDa monomeric protein on SDS ⁄ PAGE, respectively, were assayed for their specif- icity by western blotting. AbTE-D antibodies were used to rule out any nonspecific binding that might have occurred with AbTE-N because of the aggregated nature of the native protein. The purified enzyme was run on SDS ⁄ PAGE, and, after electroblotting on to nitrocellulose membrane, it was probed with AbTE-N and AbTE-D (Fig. 2C). Both antibodies identified the 22-kDa band corresponding to the monomer of thio- esterase enzyme on denaturing gel. Alcaligenes was grown to mid-exponential phase, and, after several dehydration steps, embedded in LR White resin, which was then dehydrated in several steps. Optimal ultrastructural preservation required inclusion of 0.2% glutaraldehyde in the fixative; the reactivity of the antibody was not affected by glutaral- dehyde fixation. Thin sections cut using an ultramicro- tome were incubated with primary antibodies followed by nanogold labeled secondary antibody as described in Experimental procedures and visualized under the transmission electron microscope. Fraction No A B C 0 5 10 15 20 25 30 A mn 082 A mn 082 A mn 082 0.0 0.1 0.2 0.3 0.4 0.5 0.6 L m/lomn 0 100 200 300 400 500 600 700 800 Fraction No 51015202530 0.0 0.1 0.2 0.3 0.4 Lm/nim/lo m n 0 100 200 300 400 500 600 Fraction No 20 30 40 50 60 70 0.0 0.1 0.2 0.3 0.4 0.5 0.6 a b c Fig. 1. Elution profile of thioesterase on gel filtration chromatogra- phy. (A) 13 · 425 mm Sephacryl S-300 column; (B) 13 · 290 mm Sepharose S-4B column. The column was pre-equilibrated with 100 m M Tris ⁄ HCl buffer, pH 7.6, containing 150 mM NaCl at a flow rate of mLÆh )1 . Then 250 lg protein was loaded and eluted in the same buffer. Fractions of 2.0 mL each were collected and assayed for thioesterase activity as described in Experimental procedures. (d) activity units. (C) 13 · 290 mm Sepharose S-4B column, flow rate 10 mLÆh )1 . Peak A, Blue dextran; B, thioesterase; C, thyroglobulin. Thioesterase of Alcaligenes faecalis P. Shahi et al. 2376 FEBS Journal 273 (2006) 2374–2387 ª 2006 IMTECH Different fields were observed, and the gold particles were found to be exclusively present on the surface of the cells. The immunogold labeling was much more abundant for cells where AbTE-N (Fig. 4A,B) was used as primary antibody than for those where AbTE-D (Fig. 4C) was used. No labeling occurred in the control cells where primary antibodies were derived from pre- immunized serum (Fig. 4D). Does A. faecalis have multiple thioesterases? Total thioesterase activity could not be extracted from the cells with NaCl even after several treatments. To find the presence of any other thioesterase, 10 g cells, suspended in phosphate buffer, pH 7.0 (total volume 25 mL) were sonicated in three batches of equal volume and fractionated into soluble and particulate fractions by ultracentrifugation (100 000 g for 4 h). Most of the thioesterase activity (> 80%) was present in the soluble fraction, but the particulate fraction was also found to be active. SDS ⁄ PAGE of both these fractions, run under reducing conditions showed the presence of a 22-kDa protein (Fig. 2B). The particulate fraction on incubation with 1 mL phosphate buffer, pH 7.0, con- taining 1 m NaCl, released most of the activity. Total proteins were combined and, after removal of particu- lates, were concentrated to 2.5 mL by ultrafiltration (3-kDa membrane; filtrate was devoid of thioesterase activity). Then 1 mL of the concentrated protein was applied to a Sephacryl S-300 gel-filtration column (13 · 530 mm), pre-equilibrated with Tris ⁄ HCl buffer, pH 7.5, containing 150 mm NaCl at a flow rate of 24 mLÆh )1 and eluted in the same buffer. Fractions of 1.0 mL volume were collected and assayed, but thioest- erase activity was detected only in the void volume. SDS ⁄ PAGE of the void volume under reducing condi- tions showed the presence of only 22-kDa protein. Thus, we could identify only one thioesterase activity in A. faecalis, in contrast with E. coli and Rhodopseudo- monas sphaeroides, each of which contained two thio- esterases; in addition, a third one has been implicated in E. coli [2]. Mass spectrometry Tandem MS was performed by Midwest Bio Services (Overland Park, KS, USA) on an LCQ Deca XP Plus ion trap mass spectrometer (ThermoFinnigan, Arcade, NY, USA). SDS ⁄ PAGE of purified protein was run under reducing conditions. The thioesterase band at 22 kDa from the Coomassie-stained gel was excised and subjected to in-gel trypsinization. The resulting peptide mixture was concentrated on a peptide trap col- umn and washed to remove salts and other impurities. The peptides were separated on a microcapillary C18 reverse-phase chromatography column, and the eluted peptides sprayed directly into the mass spectrometer. MS ⁄ MS spectral data were obtained and analyzed by comparing them with the NCBI nonredundant protein sequence database using turbosequest and peeks online software (http://www.bioinformaticssolutions. com:8080/peaksonline/). The observed MS ⁄ MS spectra did not match any peptide from E. coli thioesterases or any other thioesterase in the database. The following 14.2 20.1 24.0 29.0 36.0 45.0 66.0 66.0 45.0 36.0 29.0 24.0 20.1 14.2 kDa kDa 2 1 1 3 2 1 2 3 A B C Fig. 2. Purification of thioesterase, fraction- ation of thioesterase activity and western blot with antibodies to thioesterase. (A) Pro- tein samples run on 12.5% SDS ⁄ PAGE after purification. Lane 1, molecular mass marker; lane 2, purified enzyme. (B) Fractionation of thioesterase. The particulate and soluble fractions, obtained by ultracentrifugation of sonicated cells, were run on gel. Lane 1, purified thioesterase; lane 2, membrane fraction; lane 3, soluble fraction. (C) West- ern blotting. Lane 1, marker; lane 2, antibod- ies raised against purified protein (AbTE-N); lane 3, antibodies raised against gel purified and denatured protein (AbTE-D) were used as primary antibody followed by horseradish peroxidase-conjugated anti-rabbit IgG. P. Shahi et al. Thioesterase of Alcaligenes faecalis FEBS Journal 273 (2006) 2374–2387 ª 2006 IMTECH 2377 peptide fragments were obtained: YYDDNIWIAL DYCDYYQLTHKPASLEK, LTKDAKYLEKAKET YAWTK, AKETYA + WTKKHLCDPTDHLYWD NINLKGKVSK, VSKEKYAYNSGQMIQAGVLLY EETGD + EQYLR, which showed identity with a putative hydrolase from Bacteroides thetaiotaomicron VPI-5482 (accession gi|29339943|gb|AAO77738.1|) [18]. Attempts at preparation of thioesterase null mutants by chemical mutagenesis An attempt was made to obtain thioesterase null mutant by N-methyl-N¢-nitrosoguanidine mutagenesis using the standard protocol [19], but without any success. Approximately 10 000 colonies were screened in one set for any mutation in the gene encoding thioesterase enzyme with the help of plate assay [20]. The mutants giving negative or ambiguous thioesterase assay results were further analyzed for thioesterase activity by the 5,5¢-dithio-bis-(2-nitrobenzoic acid) (DTNB) method. No mutant could be found that lacked thioesterase activity. Substrate specificity of thioesterase Thioesterase-catalyzed hydrolysis of acyl-CoA derivatives The effect of chain length on the activity of thio- esterase was studied. The initial rate of hydrolysis of a series of saturated acyl-CoA derivatives at different concentrations in the range 1.5–60 lm by thioesterase (0.2 lg) was determined. At saturating concentrations, stearoyl-CoA (C 18:0 ) was the most active substrate, with the rate of hydrolysis decreasing with decreasing chain length. The acyl-CoAs of chain length longer than C 18 , were not studied. The rates of hydrolysis of palmitoyl-CoA (C 16:0 ) and myristoyl-CoA (C 14:0 )at saturating concentrations were similar. The enzyme showed very little activity towards octa- noyl-CoA, which required a higher concentration of enzyme for detectable activity. No activity was observed with acyl-CoAs having chain length smaller than C 8 . The thioesterase also possessed activity towards unsaturated long-chain acyl-CoA derivatives. V max and K m values were determined by least-squares analysis of double-reciprocal plots of the data obtained from the corresponding Michaelis–Menten plots. V max and K m values were in the range 3.58–9.73 lmolÆmin )1 Æ(mg protein) )1 and 2.66–4.11 lm, respect- ively (Table 1). Thioesterase-catalyzed hydrolysis of p-nitrophenyl esters The assay mixture (1 mL) consisted of 400 lm p-nitro- phenyl derivative in 0.1 m phosphate buffer, pH 7.2, containing 0.1 m NaCl. The reaction was started by the addition of 0.2 lg thioesterase. Initial rates were determined by measuring the increase in A 346 (e ¼ 4800 m )1 Æcm )1 ), the isosbestic point of the p-nitrophe- nol ⁄ p-nitrophenoxide couple, as described [21]. The effect of the chain length of the p-nitrophenyl esters on the activity of thioesterase was studied (Table 2). p-Nitrophenyl propanoate (C 3:0 ) was found to be the most active substrate, with the activity decreasing sharply with increasing or decreasing chain length. Diameter ( nm ) 10 15 20 25 30 35 40 selcitraP fo rebmuN 0 10 20 30 40 50 A B Fig. 3. TEM. The purified thioesterase was desalted and concentra- ted by repeated ultrafiltration using a Centricon 50-kDa membrane and suspended in water at a concentration of 600 lgÆmL )1 .TEM was performed on the carbon grid using 2% aqueous uranyl acet- ate and 2% phosphotungstic acid at pH 8.0. (A) Electron micro- graph showing granular particles with mean diameter 21.6 nm. (B) Size distribution of the thioesterase particles in TEM. Particle size distribution was evaluated by measuring the diameter of 100 parti- cles. The diameter was the mean of two right angled axes. Thioesterase of Alcaligenes faecalis P. Shahi et al. 2378 FEBS Journal 273 (2006) 2374–2387 ª 2006 IMTECH p-Nitrophenyl acetate (C 2:0 ) and p-nitrophenyl hexano- ate (C 6:0 ) were 30% as active as their (C 3:0 ) counter- part. p-Nitrophenyl dodecanoate (C 12:0 ) showed less than 10% of the maximum activity, whereas p-nitro- phenyl esters with chain length of more than C 12 were not hydrolyzed by the thioesterase. p-Nitrophenyl pro- panoate was a threefold more active substrate than the best acyl-CoA substrate, stearoyl-CoA. It is interesting to note that chain length specificity of Alcaligenes thio- esterase for p-nitrophenyl esters was opposite to that for acyl-CoA derivatives. The thioesterase activity and p-nitrophenyl esterase activities appear to be co-resident for the following reasons. (a) Diethyl pyrocarbonate completely inhib- ited both activities. (b) p-Nitrophenyl propanoate provided protection against inhibition of thioesterase activity by diethyl pyrocarbonate to the extent of 74%. The experiment was performed as follows. To a pre- cooled solution of enzyme (100 lg) in 1 mL phosphate buffer, pH 6.0, was added 50 lm p-nitrophenyl prop- ionate, immediately followed by 1 mm diethyl pyrocar- bonate (from 1 m ethanol stock solution). A parallel experiment was run as a control in which the addition of substrate was omitted. The sample was incubated for 5 min, and a 5-lL aliquot of each sample was withdrawn and assayed for activity by the DTNB method using stearoyl-CoA as substrate. (c) In an analogous manner, when stearoyl-CoA was used as AB CD Fig. 4. Transmission electron micrographs of immunogold-labeled Alcaligenes treated with various primary antibodies. Alcaligenes cells were embedded in LR white resin as described in Experimental procedures. Thin sections were incubated with primary anti- body raised against thioesterase, followed by anti-rabbit IgG with conjugated nanogold particles, and samples were analyzed under the electron microscope. Different fields were viewed. Arrowheads denote gold parti- cles. (A) Primary antibody AbTE-N raised against purified native thioesterase. (B) Enlarged view of a single cell (bar, 200 nm). (C) Primary antibody AbTE-D, raised against a gel piece corresponding to the thioest- erase band in SDS ⁄ PAGE. (D) Control in which preimmune serum was used as pri- mary antibody. P. Shahi et al. Thioesterase of Alcaligenes faecalis FEBS Journal 273 (2006) 2374–2387 ª 2006 IMTECH 2379 protecting agent and activity was assayed with p-nitro- phenyl propanoate as substrate, 93% protection against inhibition was observed. Long-chain acyl-CoAs are better substrates than aryl esters Stearoyl-CoA provided better protection against inhi- bition of thioesterase activity by diethyl pyrocarbonate than p-nitrophenyl propanoate, as shown above, which indicated that the long-chain acyl-CoAs are better sub- strates for the enzyme than aryl esters. This was fur- ther confirmed by substrate competition experiments. Aryl esterase activity against p-nitrophenyl propionate (400 lm) was inhibited by 52% when it was measured in the presence of 100 lm stearoyl-CoA, whereas the presence of 200 lm p-nitrophenyl propionate in the assay mixture of stearoyl-CoA (50 lm) had no effect on the thioesterase activity, measured by the DTNB method. Implication of a histidine residue in the active site of thioesterase The effect of various protein-modifying agents on the activity of the thioesterase was studied (Table 3). Phe- nylmethanesulfonyl fluoride, a serine-active agent, had no significant effect on the activity of the enzyme. N-Bromosuccinimide caused complete loss of activity at 1mm concentration, indicating the presence of catalyti- cally important residues such as tyrosine, tryptophan and histidine. However, dimethyl (2-hydroxy-5-nitro- benzyl)sulfonium bromide, a tryptophan-modifying agent, and N-acetylimidazole, a tyrosine inhibitor, did not have any significant effect on the thioesterase activ- ity. Diethyl pyrocarbonate, a histidine-modifying agent, caused total loss of activity at 1 mm concentration. The thiol-reactive agent DTNB did not have a significant effect on the thioesterase activity, allowing the use of DTNB in the thioesterase assay. Picrylsulfonic acid, a lysine-modifying agent, also had no effect on the activ- ity of the enzyme. These results indicate the presence of a catalytically important histidine residue in the enzyme. The presence of a histidine residue was further supported by the following experiments. (a) Reversal of inhibition by hydroxylamine. The activity of the enzyme could be partially recovered by the treatment of diethyl pyrocarbonate-inhibited enzyme with hydroxylamine. An aliquot of enzyme, inactivated with 1 mm diethyl pyrocarbonate at 4 °C, was incubated at 25 °C, for 8 h with 250 mm hydrox- ylamine and assayed for thioesterase activity by the DTNB method after extensive dialysis, as described in Experimental procedures. Its activity was expressed as a percentage of that obtained from an experiment, run in parallel, in which same amount of active enzyme (no inhibitor added) was incubated with 250 mm hydroxylamine for 8 h at 25 °C and assayed for activity in the same manner. The treatment of the diethyl pyrocarbonate-inhibited enzyme with Table 1. Thioesterase catalyzed hydrolysis of acyl-CoA derivatives. A solution of acyl-CoA derivative was prepared in 100 m M Tris ⁄ HCl buffer, pH 7.6, at various concentrations in the range 1.5–60 l M. The reaction was started by the addition of an aliquot of enzyme (0.2 lg), and initial rates of hydrolysis were measured by the DTNB method as described in Experimental procedures. V max and K m val- ues were determined by least-squares analysis of double-reciprocal plots of the data obtained from the corresponding Michaelis– Menten plots. Sr. No., serial number. Sr. No. Substrate K m (lM) V max [lmolÆmin )1 Æ (mg protein) )1 ] 1 Lauroyl-CoA 3.48 ± 0.3 3.58 ± 0.3 2 Myristoyl-CoA 2.66 ± 0.2 6.61 ± 0.5 3 Palmitoyl-CoA 4.11 ± 0.2 7.17 ± 0.3 4 Stearoyl-CoA 3.59 ± 0.1 9.73 ± 0.2 5 Myristoleoyl-CoA 3.39 ± 0.3 4.77 ± 0.4 6 Palmitoleoyl-CoA 3.90 ± 0.1 5.57 ± 0.3 7 cis-Vaccenoyl-CoA 2.84 ± 0.3 6.05 ± 0.5 Table 2. Thioesterase-catalyzed hydrolysis of aromatic esters. The assay mixture (1 mL) consisted of 400 lM p-nitrophenyl derivative in 0.1 M phosphate buffer, pH 7.2, containing 0.1 M NaCl. The reaction was started by the addition of 0.2 lg thioesterase. Initial rates were determined by measuring the increase in A 346 (e ¼ 4800 M )1 Æcm )1 ), the isobestic point of the p-nitrophenol ⁄ p-nitrophenoxide couple. Sr. No. p-Nitrophenyl ester Specific activity [lmolÆmin )1 Æ(mg protein) )1 ] Relative activity (%) 1 p-Nitrophenyl acetate 9.16 30.14 2 p-Nitrophenyl propanoate 30.52 100 3 p-Nitrophenyl butanoate 15.87 51.02 4 p-Nitrophenyl hexanoate 9.77 33.16 5 p-Nitrophenyl dodecanoate 2.44 8.67 6 p-Nitrophenyl palmitate Not detected – 7 p-Nitrophenyl stearate Not detected – Thioesterase of Alcaligenes faecalis P. Shahi et al. 2380 FEBS Journal 273 (2006) 2374–2387 ª 2006 IMTECH hydroxylamine in this way resulted in 58.7% recovery of activity. (b) A 35.7% increase in absorption at k 245 corres- ponding to N-ethoxycarbonylation of histidine on inactivation of thioesterase with diethyl pyrocarbonate for 1 h was observed in differential spectra obtained as described in Experimental procedures. (c) Stearoyl-CoA, a substrate of the enzyme provided  93% protection against inhibition with diethyl pyro- carbonate, as shown above. Effect of temperature and pH on the activity of the thioesterase Optimum pH and pH stability For determination of the optimum pH of the enzyme, its activity was measured at various pHs ranging from 5.5 to 10.5 at 30 °C (Fig. 5A). The maximum activity of thioesterase was obtained at pH 10.5. For pH 5.5– 8.0, phosphate buffers were used, in which  80% of maximum activity was retained. For pH 7.2–9.0, Tris ⁄ HCl buffers were used. Although the trend of activity in the pH range 7.2–8.0 was the same as that in phosphate buffer, the activity was  20% less than that in phosphate buffer. For pH 9.0–10.5, sodium carbonate buffer was used. At pH 9.0, the activity was  40% less in carbonate buffer than in Tris buffer. The effects of buffer composition on enzyme activity have been reported previously [22]. These effects may be due to the effects of the buffer on the oligomeriza- tion status of the enzyme [23]. An alternative explan- ation is that the binding affinity of the enzyme for the substrate is modified, presumably because of differ- ences in the interaction of the buffer ions with the binding site [24]. Activity at pH 10.5 was maximum and set as 100%. Activity above pH 10.5 was not stud- ied. Controls were used in each case to compensate for chemical hydrolysis, which was substantial at higher pH. Although maximum activity was obtained at pH 10.5, all studies were carried out at pH 7.0–7.5 as the substrates are prone to degradation under basic conditions. To evaluate pH stability, the enzyme was incubated in different buffers, pH 5.5–10.5, at 30 °C for 20 h. The remaining activity is expressed as a percentage of the activity relative to the activity in the corresponding buffer at time zero. Thioesterase retained almost Table 3. Effect of protein-modifying reagents on thioesterase activ- ity. Purified and dialyzed thioesterase at a concentration of 20 lgÆmL )1 was incubated with each reagent at 25 °C for 15 min and dialyzed against 50 m M Tris ⁄ HCl buffer, pH 7.5, at 4 °C with four buffer changes for 12 h. Residual activity, percentage of the original activity, was calculated by the DTNB method as described in Experimental procedures. Sr. No. Reagent (1 m M) Residual activity (%) 1 N-Bromosuccinimide 0.0 2 Phenylmethanesulfonyl fluoride 90.7 3 N-Acetylimidazole 90.7 4 Iodoacetamide 98.9 5 Diethyl pyrocarbonate 0.0 6 Dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide 89.0 7 Picrylsulfonic acid 89.0 8 p-Chloromercuribenzoate 88.3 95,5¢-Dithiobis-(2-nitrobenzoic acid) 98.4 10 Dithiothreitol 80.0 11 None 100 pH 567891011 )%( ytivitca laudiseR 0 20 40 60 80 100 120 pH 567891011 )%( ytivitcA evitaleR 20 40 60 80 100 120 A B Fig. 5. (A) Effect of pH on the activity of thioesterase. Assays were performed at 30 °C in various buffers at different pH. The activity in carbonate buffer at pH 10.5 was set as 100%, all other values are relative to it. (B) pH stability of thioesterase. A predetermined amount of thioesterase was incubated in different buffers for 20 h at 30 °C and assayed for thioesterase activity. The remaining activ- ity is expressed as percentage of activity relative to the activity in the corresponding buffer at time zero. (d) phosphate buffer; (.) Tris ⁄ HCl buffer; (n) carbonate buffer. P. Shahi et al. Thioesterase of Alcaligenes faecalis FEBS Journal 273 (2006) 2374–2387 ª 2006 IMTECH 2381 complete activity at pH 5.5–6.0 (Fig. 5B). It retained  80% activity at pH 7.0–7.5 and was relatively unsta- ble under alkaline conditions. After 20 h incubation at pH 10.5 in carbonate buffer, only 20% activity was retained. Thermal properties of the thioesterase Thioesterase activity was determined at different tem- peratures in phosphate buffer at pH 7.0. Maximum thioesterase activity occurred at 65 °C (Fig. 6A). About 60–80% of maximum activity was retained at temperatures of 25–70 °C. There was sharp decline in activity at 75–80 °C; the enzyme retained  20% of maximum activity at 80 °C. To evaluate temperature stability, enzyme in phos- phate buffer, pH 7.0 was incubated for 3 h at different temperatures. Thioesterase activity was assayed at 30 °C by the DTNB method as described in Experi- mental procedures. The activity at zero time at 30 °C is assumed to be 100%, and all other values are expressed relative to it. Most of the enzyme activity was retained at 25–50 °C (Fig. 6B). After 3 h, 70% of the activity remained at 70 °C. At 80 °C, most of the activity was lost after 3 h. Effect of metal ions on thioesterase activity The effect of various bivalent metal ions on thioest- erase activity was studied by the DTNB method as described in Experimental procedures. Zn 2+ showed concentration-dependent partial inhibition of the enzyme. It had no effect on the activity at 1 mm con- centration but caused 40% inhibition at 10 mm.Hg 2+ and Cu 2+ caused complete inhibition of enzyme activ- ity at 1 mm concentration. The activity was enhanced by 10–20% when enzyme assays were performed in the presence of Mg 2+ ,Ni 2+ or Co 2+ . Effect of temperature on the kinetics of thioesterase-catalyzed hydrolysis of palmitoyl-CoA, palmitoleoyl-CoA and cis-vaccenoyl-CoA The cells were grown at 25, 30 and 37 °C and assayed for thioesterase activity by the DTNB method. No difference in activity was observed, ruling out a tem- perature-dependent change in expression levels of thio- esterase. The kinetics of thioesterase activity was determined at 20 and 37 °C with palmitoyl-CoA, palmitoleoyl-CoA or cis-vaccenoyl-CoA as substrate (Table 4). With palmitoyl-CoA as substrate, thioest- erase showed only marginal changes in V max and K m values at both the temperatures. However, approxi- mately twofold reduced affinity and catalytic efficiency was observed when palmitoleoyl-CoA or cis-vaccenoyl- CoA was the substrate at the lower temperature. Discussion Two thioesterases, I and II, that cleave acyl-CoA mol- ecules in vitro have been characterized from E. coli. Thioesterase I is a periplasmic protein of 20.5 kDa and has an active site similar to serine proteases [2,9]. Thio- esterase II is a tetrameric protein with identical subunits of 32 kDa and is insensitive to inhibition with di-isopropyl fluorophosphate. A histidine residue in thioesterase II has been implicated in the cleavage of the thioester bond [10,11]. In comparison, thioesterase from A. faecalis exists as large homomeric granular aggregates (21.6 nm average diameter) of 22-kDa sub- units (Figs 2A and 3). Phenylmethanesulfonyl fluoride, a serine-active reagent, failed to inhibit the catalytic activity of thioesterase. A. faecalis thioesterase was digested with trypsin, and the resulting peptides separ- ated on a microcapillary C18 reverse-phase chromato- graphy column. MS ⁄ MS data were obtained and analyzed by comparing them with the NCBI non- redundant protein sequence database. The observed Temperature ( o C) 20 30 40 50 60 70 80 90 )%( ytivitca laudiseR/evitaleR 0 20 40 60 80 100 120 A B Fig. 6. Thermal properties of thioesterase. (A) Optimum tempera- ture of thioesterase activity. Assays were performed in 50 m M phosphate buffer, pH 7.0. The relative activity is expressed as per- centage of maximum activity attained under the experimental conditions. (B) Thermostability of thioesterase. A predetermined amount of thioesterase was incubated for 3 h at different tempera- tures and then assayed for thioesterase activity in 50 m M phos- phate buffer at 30 °C. The activity at zero time at 30 °C is assumed to be 100%; all other values are expressed relative to it. Thioesterase of Alcaligenes faecalis P. Shahi et al. 2382 FEBS Journal 273 (2006) 2374–2387 ª 2006 IMTECH MS ⁄ MS spectra did not match any peptide from E. coli thioesterases or any other thioesterase in the database. Thioesterase was found to be associated exclusively with the surface of cells as revealed by ultrastructural studies using electron microscopic im- munogold labeling studies (Fig. 4). A histidine residue has been implicated in the active site of the enzyme based on the following observations. (a) Incubation of thioesterase with 1mm diethyl pyrocarbonate resulted in complete loss of enzyme activity. (b) An increase in absorption at k 245 corresponding to N-ethoxycarbonylation of histi- dine on inactivation with diethyl pyrocarbonate was observed when differential spectra were recorded. (c) Approximately 60% of the enzyme activity could be recovered by the treatment of diethyl pyrocarbo- nate-inhibited enzyme with hydroxylamine. (d) Stea- royl-CoA, a substrate of the thioesterase, provided  93% protection against inactivation by diethyl pyrocarbonate. Alcaligenes thioesterase was active in and stable to a wide range of temperatures and pH values. Maximum activity was observed at 65 °C and pH 10.5 and varied between 60% and 80% at 25–70 °C and pH 6.5–10 (Figs 5 and 6). Enzyme activity remained unaltered after incubation in phosphate buffer for 3 h at 50 °C. Thioesterase hydrolyzed saturated and unsaturated acyl-CoAs of C 10 to C 18 chain length with V max and K m values in the range 3.58–9.73 lmolÆmin )1 Æ(mg pro- tein) )1 and 2.66–4.11 lm, respectively (Table 1). At saturating concentrations, stearoyl-CoA (C 18:0 ) was the most active substrate, with the rate of hydrolysis decreasing with decreasing chain length. Thioesterase also has chymotrypsin-like activity and was able to hydrolyze p-nitrophenyl esters of C 2 to C 12 chain length. The most active substrate was C 3 , with the activity falling sharply with increase or decrease in chain length. Long-chain p-nitrophenyl esters were not hydrolyzed by the enzyme. The odd-chain C 3 esters are unlikely to be natural substrates of the enzyme. In any case, the substrate competition experiments clearly demonstrated that the long-chain acyl-CoAs are better substrates than p-nitrophenyl esters. The ratio of saturated ⁄ unsaturated fatty acid in mem- brane phospholipids is tightly controlled in a tempera- ture-dependent manner in micro-organisms, which allows proper thermal regulation of membrane fluidity [25–27]. Thermal regulation of membrane fluidity is common to all organisms. Lower growth temperatures result in an increase in the number of unsaturated phospholipids in the membrane. E. coli can synthesize phospholipids almost entirely from exogenous fatty acids supplied by the growth medium. The satur- ated ⁄ unsaturated fatty acids in membrane phospho- lipids, synthesized from exogenous fatty acids is similar to de novo ratio in a temperature-controlled fashion [28]. A site for thermal regulation must therefore exist at the level of utilization of exogenous fatty acids, in addi- tion to a well-defined site for thermal regulation in de novo fatty acid synthesis [26]. Previous literature sug- gests that such a regulation is likely to be at the enzyme and not gene level [29]. Starting from exogenous fatty acids, the incorporation is known to involve first the formation of acyl-CoA derivatives. Therefore, the possi- bility exists that a thioesterase may be involved in this thermal regulation, if it is able to control the ratios of saturated and unsaturated fatty acyl-CoAs in a tem- perature-dependent manner. V max ⁄ K m values for palmi- toyl-CoA were 1.74 and 1.57 at 37 and 20 °C, respectively (Table 4). The corresponding values for palmitoleoyl-CoA were 1.43 and 0.64, and 2.13 and 0.92 for cis-vaccenoyl-CoA. The K m values for palmitoyl- CoA at 37 and 20 °C were 4.11 and 3.85, respectively, whereas the corresponding values for palmitoleoyl-CoA were 3.90 and 7.20, and 2.84 and 5.51 for cis-vaccenoyl- CoA. Whereas the affinity and catalytic efficiency of Al- caligenes thioesterase were reduced by about twofold for palmitoleoyl-CoA and cis-vaccenoyl-CoA at lower temperature, these remained largely unaltered for palmi- toyl-CoA, which should result in a higher ratio of unsat- urated ⁄ saturated fatty acyl-CoA at lower temperature compared with higher temperature. In principle, the Table 4. Effect of temperature on the kinetics of thioesterase-catalyzed hydrolysis of palmitoyl-CoA, palmitoleoyl-CoA and cis-vaccenoyl- CoA. A solution of acyl-CoA derivative was prepared in 100 m M Tris ⁄ HCl buffer, pH 7.6, at various concentrations in the range 1.5–60 lM. The reaction was started by the addition of an aliquot of enzyme (0.2 lg), and initial rates of hydrolysis were measured by the DTNB method as described in Experimental procedures. V max and K m values were determined by least-squares analysis of double-reciprocal plots of the data obtained from the corresponding Michaelis–Menten plots. Temp (°C) Palmitoyl-CoA Palmitoleoyl-CoA cis-Vaccenoyl-CoA K m V max V max ⁄ K m K m V max V max ⁄ K m K m V max V max ⁄ K m 20 3.85 6.05 1.57 7.20 4.60 0.64 5.51 5.07 0.92 37 4.11 7.17 1.74 3.90 5.57 1.43 2.84 6.05 2.13 P. Shahi et al. Thioesterase of Alcaligenes faecalis FEBS Journal 273 (2006) 2374–2387 ª 2006 IMTECH 2383 [...]... voltage 60–80 kV), and random fields were photographed The prints of the micrographs were made at the desired magnification for further analysis Controls included the labeling of each set of samples with preimmune serum (i.e normal rabbit serum) instead of anti -thioesterase serum Enzyme-catalyzed hydrolysis of acyl-CoA derivatives Thioesterase- catalyzed hydrolysis of various long-chain acyl-CoAs was studied.. .Thioesterase of Alcaligenes faecalis reduced affinity and decreased catalytic efficiency of thioesterase for unsaturated fatty acyl-CoA at lower temperature compared with higher temperature may account for the temperature-dependent regulation of membrane phospholipids A similar observation has been made to define the probable mechanisms by which temperature fixes the limit of growth for the... Sabri A, Bare G, Jacques P, Jabrane A, Ongena M, Van Heugen JC, Devreese B & Thonart P (2001) Influ- FEBS Journal 273 (2006) 2374–2387 ª 2006 IMTECH Thioesterase of Alcaligenes faecalis ence of moderate temperatures on myristoyl-CoA metabolism and acyl-CoA thioesterase activity in the psychrophilic antarctic yeast Rhodotorula aurantiaca J Biol Chem 276, 12691–12696 31 Holt JG, Krieg NR, Sneath PHA, Staley... yeast, Rhodotorula aurantiaca A1 9, isolated from Antarctic ice, which is unable to grow at moderate temperatures (> 20 °C) [30] A faecalis provides an interesting system to explore such a possibility Experimental procedures Materials Medium components, peptones, beef extract, agar powder, etc were purchased from Himedia Laboratories Pvt Ltd (Bombay, India) Metal salts, NaCl and buffer reagents were from. .. reaction was started by the addition of an aliquot of enzyme (0.2 lg) to a final volume of 0.5 mL The contents were incubated at 37 °C, and change in absorbance was measured at 1 min interval for 5 min TEM of thioesterase Protein eluted from a Sepharose CL-4B column was dialyzed against water and concentrated by ultrafiltration using 50-kDa Centricon filters Protein samples were analyzed by negative-staining... in thioesterase activity, was used in these studies Assay of thioesterase activity Thioesterase activity was measured by following the increase in A4 12 (e ¼ 7684 m)1Æcm)1), when free CoASH 2384 P Shahi et al generated during deacylation of acyl-CoA reacted with DTNB as previously described [32] In brief, each assay contained 100 mm Tris ⁄ HCl, pH 7.6, 0.4 mm DTNB and 100 lm acyl-CoA The reaction was... thioesterase B gene associated with hormonal induction of peroxisome proliferation J Biol Chem 268, 14278–14284 5 Loader NM, Woolner EM, Hellyer A, Slabas AR & Safford R (1993) Isolation and characterization of two Brassica napus embryo acyl-ACP thioesterase cDNA clones Plant Mol Biol 23, 769–778 6 Naggert J, Witkowski A, Wessa B & Smith S (1991) Expression in Escherichia coli, purification and characterization. .. Bacterial strain The strain isolated from soil samples was identified as a bacterium, A faecalis according to Bergey’s Manual [31], and was designated A faecalis ISH108 The strain has been deposited with Microbial Type Culture Collection, MTCC (Institute of Microbial Technology, Chandigarh, India) (http://mtcc.imtech.res.in/cgi-bin/mainhit.pl, accession number MTCC7733) Cell growth and protein extraction... 199–205 Appleton & Lange, San Mateo, CA 9 Barnes EM Jr & Wakil SJ (1968) Studies on the mechanism of fatty acid synthesis XIX Preparation and general properties of palmitoyl thioesterase J Biol Chem 243, 2955–2962 10 Bonner WM & Bloch K (1972) Purification and properties of fatty acyl thioesterase I from Escherichia coli J Biol Chem 247, 3123–3133 11 Naggert J, Narasimhan ML, DeVeaux L, Cho H, Randhawa ZI,... characterization of two mammalian thioesterases involved in fatty acid synthesis Biochem J 273, 787–790 7 Naggert J, Williams B, Cashman DP & Smith S (1987) Cloning and sequencing of the medium-chain S-acyl fatty acid synthetase thioester hydrolase cDNA from rat mammary gland Biochem J 243, 597–601 8 Mayes PA (1990) Biosynthesis of fatty acids In Harpers Biochemistry, 22nd edn (Murray RK, Granner DK, Mayes PA & . Characterization of a novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis Puja Shahi* † , Ish Kumar* ‡ , Ritu Sharma, Shefali Sanger and. 2006) doi:10.1111/j.1742-4658.2006.05244.x A novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis has been isolated and characterized. The protein was extracted from the cells with