Báo cáo khoa học: Expression and characterization of the protein Rv1399c from Mycobacterium tuberculosis A novel carboxyl esterase structurally related to the HSL family docx

Expression and characterization of the protein Rv1399c from Mycobacterium tuberculosis A novel carboxyl esterase structurally related to the HSL family Ste ´ phane Canaan 1 , Damien Maurin 1 , Henri Chahinian 2 ,Be ´ ne ´ dicte Pouilly 1 ,Ce ´ cile Durousseau 1 , Fre ´ de ´ ric Frassinetti 1 ,Lore ´ na Scappuccini-Calvo 1 , Christian Cambillau 1 and Yves Bourne 1 1 Architecture et Fonction des Macromole ´ cules Biologiques, AFMB UMR 6098, CNRS, Marseille, France; 2 Laboratoire d’Enzymologie Interfaciale et de Physiologie de la Lipolyse, CNRS UPR 9025, Marseille, France The Mycobacterium tuberculosis genome contains an unusually high number of proteins involved in the metabo- lism of lipids belonging t o the Lip family, includ ing various nonlipolytic and lipolytic hydrolases. Driven by a structural genomic approach, w e have bio chemically characterized t he Rv1399c gene product, LipH, previously annotated as a putative lipase. Rv1399c was overexpressed in E. coli as inclusion bodies and r efolded. Rv1399c efficiently hydro- lyzes soluble triacylglycerols and vinyl esters. It is inactive against emulsified substrate and its catalytic activity is strongly inhibited by the diethyl p aranitrophenyl phosphate (E600). These kinetic behaviors unambiguously classify Rv1399c as a nonlipolytic rather than a lipolytic hydrolase. Sequence alignment reveals that this enzyme b elongs to the a/b hydrolase fold family and shares 30–40% amino acid sequence identity w ith members of the hormone-sensitive lipase subfamily. A model of Rv1399c derived from homologous three-dimensional structures reveals a canon- ical catalytic triad (Ser162, His290 and Asp260) located at the bottom o f a solvent accessible pocket lined by neutral or charged residues. Based on this m odel, kinetic data o f the Arg213Ala mutant partially explain the role of the guanid- inium moiety, located close to His290, to confer an unusual low pH s hift of the catalytic histidine in the wild type enzyme. Overall, these data identify Rv1399c as a new nonlipolytic hydrolase from M. t uberc ulosis and we thus propose to reannotate its gene product as NLH-H. Keywords: e sterase; nonlipolytic hydrolase; Rv1399c; tuber- culosis. The recent elucidation of the complete sequence of Myco- bacterium tuberculosis genome [1] has offered new perspec- tives for the search of novel drugs against tuberculosis. The disease was responsible for around 2.5–3 million deaths in 2002 and t he World Health Organization (WHO) estimates 8 million new tuberculosis patients each year [2]. The current genome annotation consists of  4000 predicted proteins, classified into 11 distinct protein groups [3], of which 48% have unknown function. Comparative sequence analysis of the M. tuberculosis genome has revealed that it contains 250 enzymes involved in lipid metabolism com- pared to only 50 in Escherichia coli. Among these enzymes, a family of 21 carboxyl este r h ydrolases, called Lip (A to W, except K and S), have been annotated as putative esterases or lipases, based on the presence of the consensus sequence GXSXG characteristic of members of the a/b hydrolase fold family. Within this family, the recent crystal structure of the M. tuberculosis antigen 85C (Ag85C) [4], a mycolyl- transferase required for survival of mycobacteria, along with that of the noncatalytic M. t uberculo sis MPT51 protein ( FbpC1) [5], which is involved i n mycobacteria pathogenicity, have revealed that they share the same a/b hydrolase fold. Therefore, a detailed biochemical charac- terization of all members of the Lip family should be performed beyond the computational analysis. For many years, it was generally assumed that lipases are poorly active against soluble esters a nd become mark edly active when the solubility limit of the substrate is exceeded, a phenomenon called interfacial activation [6]. In contrast, esterases do not share this behavior and exhibit their maximal activity on esters in solution. Unfortunately, biochemical studies of several lipases have showed that the interfacial activation phenomenon cannot be considered a general (and sufficient) rule to discriminate between a lipase and esterase [7–9]. A clear d istinction between lipases and este rases was estab- lished recently from the comparison of the K 1/2 values of these two classes o f carboxyl ester hydrolas es using p artially soluble triacylglycerols and vinyl esters as substrates [7]. Where a lipase can also be considered an esterase, the K 1/2 values represent a reliable criterion to discriminate between these two classes of enzymes. Consequently, we propose to rename lipases as lipolytic hydrolases (LH) while the remaining members of th e family denote nonlipolytic hydro- lases (NLH). Therefore, without a detailed biochemical Correspondence to S. Canaan, A rchitecture et F onction des Macro- mole ´ cules Biologiques, AFMB UMR 6098, CNR S, 3 1 C hemin J oseph Aiguier, 13402 Marseille Cedex 20, France. Fax: +33 491 16 45 36, Tel.: +33 491 16 45 12, E-mail: stephane.canaan@ibsm.cnrs-mrs.fr Abbreviations : HSL, h ormone-sensitive lipase; NLH, nonlipolytic hydrolase; LH, lipolyt ic hydrolase; IB, inclusion bodies; IPTG, iso- propyl thio-b- D -galactoside; CMC, c ritical m icellar c oncentration; E600, diethyl para-nitrophenyl phosphate; D LS , dynamic light scattering. (Received 7 June 2004, accepted 17 August 2004) Eur. J. Biochem. 271, 3953–3961 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04335.x characterization, the Lip term could be c onfused to refer to a NLH, and the original accession number should b e used to avoid confusion. Here we report the cloning, expression and refolding of the M. tuberculosis Rv1399c gene product, a member o f the Lip family. The biochemical characterization of Rv1399c, using triacylglycerols and vinyl esters as substrates, demon- strates that this carboxyl ester hydrolase must be classified as an NLH instead of an LH as proposed initially by bioinformatic tools [1,3]. Rv1399c efficiently hydrolyzes short-chain t riacylglycerols and vinyl esters and has no detectable activity against emulsified substrates. We thus propose to rename Rv1399c NLH-H instead of LipH. Sequence alignment has revealed that this enzyme defines a new NLH that is structurally related to the hormone- sensitive lipase (HSL) family and a model derived from a subset of homologous 3D structures reveals the architecture and functionalities of the Rv1399c active site, consistent with our experimental data. Based on this model, the kinetic behavior of the Arg213Ala mutant partially explains the unusual acidic pKa shift observed f or the catalytic His290 residue in the wild-type (wt)enzyme. Experimental procedures Materials The Pfx DNA polymerase, pDonor 201, pET-Dest42 and pDest17 plasmid vectors were purchased from Invitrogen. BL21(DE3)pLysS E. coli cells were purchased from Nov- agen. N i + -agarose gel was obtained from Amersham Biosciences. Vinyl acetate, vinyl propionate, vinyl butyrate, triacetin, t ributyrin and the diethyl para-nitrophenyl phos- phate (E600) were obtained from Sigma–Aldrich–Fluka. Tripropionin was purchased from Acros Organics (Geel, Belgium). Methods Cloning. The cDNA fragment of the Rv1399c ORF was amplified by PCR from the MTCY21B4.16c cosmid provided by the Pasteur Institut [1,10]. The primers, containing, at their 5¢-and3¢-ends, the respective attB1 and attB2 recombination sites were: 5¢-taacagagccg accgtcgcccgg-3¢ (forward primer) and 5¢-cttatgcgtgcaa cgccctctt-3¢ (reverse primer). The PCR product was purified from agarose gel and was inserted into t he expression vector following the manufacturer’s instruction (Gateway, Invi- trogen). The correct insertion of the Rv1399c ORF was confirmed by DNA sequencing. In our hands, protein expression using the commercially available pDest 17 plasmid frequently appeared to be constitutive and occurred whether or not the isopropyl t hio- b- D -galactoside (IPTG) inducer was added to the medium. This problem, which was unsolved using the BL21(DE3)- pLysS cells known to over-express lysozyme as a compet- itive inhibitor of the T7 RNA polymerase, was most probably due to the absence of the lac operator dowstream of the T7 p romoter. To overcome this problem, a derivative of the pDest 17 plasmid, pD est 1 7O/I, was constructed from the pET-Dest42 plasmid and contains a P shAI-XbaI- digested fragment that encompassed the Lac I gene (under the control of a constitutive promoter) upstream the T7 promoter followed by Lac O (the DNA binding site of Lac I). The resulting construct constitutively expresses Lac I, which inhibits the T7 RNA polymerase in b inding to its specific DNA binding site on Lac O. Mutagenesis. Site-directed mutagenegis, which was per- formed using the QuickchangeÒ site-directed mutagenesis system (Stratagen, La Jolla, CA, USA), was used for the mutation of Arg213 fi Ala. The oligonucleotides u sed were: 5¢-gcgccaatcctggacgctgacgtcatcgacgcg-3¢ (forward) and 5¢-cgcgtcgatgacgtcagcgtccaggattggcgc-3¢ (reverse). The bases in bold indicate the location of the mutation. DNA sequence of the mutant was confirmed by DNA sequencing (Millegen, Prologue Biotech, France). Protein expression. BL21(DE3)pLysS cells were trans- formed with the expression construct pDest 17O/I harbor- ing the Rv1399c coding region. Cells were grown at 37 °C in LB medium containing 100 lgÆmL )1 ampicillin and 34 lgÆmL )1 chloramphenicol until a D 600 value between 0.6 and 0.8 was reached. Protein expression w as induced by adding 2 m M IPTG for 4 h at 37 °Candthen16hat15°C. Rv1399c was highly overexpressed but found exclusively in inclusion bodies. Purification of inclusion bodies. Cells were harvested at 4 °C by centrifugation at 9000 g for 15 min. The pellet was resuspended in ice-cold lysis buffer [50 m M Tris/HCl, pH 8.0, 150 m M NaCl, 10 m M imidazole, 1 m M EDTA, 0.1% (v/v) Triton X-100, 0.25 mgÆmL )1 of lysozyme and 1m M phenylmethanesulfonyl fluoride] and stored at )80 °C overnight. T he pellet was thawed on ice for 1 h andthen10lgÆmL )1 DNase a nd 20 m M MgSO 4 (final concentration) were added to the cell s uspension f or 30 min. Cells were disrupted by ultrasonication (10· with a 15 s cycle) using a Branson Sonifier 450. Inclusion bodies were separated from t he cell e xtract by centrifugation at 17 000 g for 30 m in. The pellet was then washed with 10 m M Tris/ HCl, pH 8.0 and 150 m M NaCl, sonicated (4· with a 15 s cycle) and collected by centrifugation at 17 000 g for 20 min, this procedure was repeated three times. Inclusion bodies were solubilized by stirring at 4 °C overnight in a 40 mL solution containing 10 m M Tris/HCl, pH 8.0, 150 m M NaCl and 6 M guanidine hydrochloride. The solubilized protein was clarified from insoluble material at 4 °C by centrifugation at 17 000 g for 15 min The super- natant was l oaded (3 mLÆmin )1 ) onto a Ni + -agarose column (1 mL resin for 5 mg of recombinant protein) previously equilibrated with buffer A (10 m M Tris/HCl, pH 8.0, 150 m M NaCl, 1 0 m M imidazole and 8 M urea). The column was then washed using five column volumes of 5% and 10% of buffer B (buffer A + 500 m M imidazole). Enzyme was eluted with 50% of buffer B; f ractions of eluted peaks containing purified Rv1399c were analyzed by SDS/ PAGE [11], pooled and concentrated up to 4–5 mg ÆmL )1 using an Amicon cell. Refolding. Purified Rv1399c was refolded by a dilution method consisting of diluting the concentrated protein in buffers with different pH and various compositions. The refolding c onditions were determined by a refolding method 3954 S. Canaan et al. (Eur. J. Biochem. 271) Ó FEBS 2004 based on the measurement of the turbidity at 340 nm using a 96-well plate [12]. The final refolding conditions consisted of dilut ing Rv1 399c 20· in 50 m M Trisbuffer,pH 7,at4 °C for 2 days. Rv1399c was concentrated up to 2–3 mgÆmL )1 and traces of urea and imidazole were removed using a desalting column ( HiPrep TM 26/10, Amersham Bioscienc es). Rv1399c was then concentrated up to 3 mgÆmL )1 and stored overnight at )20 °C, and after thawing, the ÔactiveÕ refolded material was recovered by centrifugation at 17 000 g for 15 min. Rv1399c is stable in the refolding buffer for at least 1 month at 4 °Candcanbestoredat )20 °C for several months. Protein concentration was deter- mined by measuring at A 280 using e 280 ¼ 1.399 mg )1 Æ mLÆcm )1 . Electrospray mass spectrometry was performed using a Voyager-DE RP spectrometer (PerSecptive, Biosys- tems) and mass analysis of trypsin-digested peptides confirmed the expected calculated molecular mass of 36 313 Da. Samples (0.7 lL containing 15 pmoles) were mixed with an equal volume of sinapinic acid matrix solution and spotted on the target, then dried at room temperature for 10 min. Circular dichoism and dynamic light scattering. The presence of secondary structural elements in refolded Rv1399c was assessed by CD using a Jasco PTC-423S apparatus and analyzed with the program CDNN ( CD SPEC- TRA DECONVOLUTION , version 2.1). Rv1399c (0.2 mgÆmL )1 ) spectra we re recorded at 20 °C between 185 and 260 nm in 10 m M Tris/HCl, pH 7.0, with a 30 s averaging step. The final CD spectrum was obtained from the average of three passes. The aggregation level of purified Rv1399c was estimated by dynamic light scattering (DLS) analysis following the manufacturer’s instructions. Experiments were performed at 20 °C with filtered (Millex syringe filters, 0.22 lm; Millipore Corp.) p rotein samples (12 lLat3mgÆmL )1 ) using a Dynapro MSTC-200 (Protein Solutions). All calculations were performed using the software provided by the manufacturer. Kinetic assays. Enzymatic hydrolysis of solutions and emulsions of various esters was followed potentiometrically at 2 5 °C using a pH-stat (TTT 80, Radiometer, Copenha- gen, Denmark) for at least 5 min. Assays were performed in 30 mL of 2.5 m M of Tris/HCl buffer, pH 7.0 and 0.1 M NaCl. Release of fatty acid was titrated with 0.1 M NaOH. Enzymatic activity was expressed in units (U) per mg of protein where 1 U corresponds to the liberation of 1 lmol acidÆmin )1 under standard conditions [13]. Assays using olive oil and vinyl laurate were performed at pH 8.5 due to the high pKa values of the oleic and l auric acids (8.1 and 7.4, respectively). pH stability – pH and temperature dependence activ- ity. Tripropionin was chosen as substrate to perform the pH and temperature dependence experiments rather than more volatile vinyl esters. The pH stability profile of Rv1399c was obtained after enzyme incubation for 1 h using 100 lL of buffers at different pH values containing, 150 m M NaCl and: 150 m M sodium acetate, pH 4 .0; 150 m M sodium acetate, pH 5.0; 150 m M Mes, pH 6.0; 150 m M Tris/HCl, pH 7.0; 150 m M Tris/HCl, pH 8.0 and 150 m M glycine, pH 9.0. The residual activity was determined potentiometrically at 25 °C at p H 7.0 using tripropionin at the concentration of 9.3 m M as substrate as above described. For the determination of the temperature dependence profile, buffer and substrate were pre-equili- brated and the background was recorded for 5 min without the enzyme. The histidine titration cu rve was performed using vinyl butyrate (20.7 m M ) as a substrate. The pH value was adjusted and the enzyme (7.4 lg) was added in the vessel. Activity assays at acidic pH were corrected using the calculated pKa value of 4.87 for butyric acid. Inhibitor assay. Purified Rv1399c (2.51 nmol) was pre- incubated at 25 °C with different E600 inhibitor/enzyme molar ratio (2 and 10) in a final v olume of 32 lL. The remaining activity was measured potentiometrically as a function of time using tripropionin as above described. Model building. The Rv1399c model was generated from the automatic protein structure homology-modeling server using SWISS - MODEL software (Biozentrum) [14–16], and validated by the PROCHECK program [17]. Sequence align- ment was performed initially using t he multiple sequence alignment s oftware T - COFFEE [18], d isplayed wi th ESPRIT [19] and then manually adjusted. The E600 inhibitor, which was taken from the crystal structure of the cutinase–E600 complex [20], was positioned into the active site of Rv1399c based on superimposed Ca atoms. Figure 4 was drawn with SPOCK [21] using the coordinates of Ag85C [4], cutinase [20,22] and Bacillus subtilis lipase A [23]. Results and Discussion Cloning and expression of Rv1399c The Rv1399c gene product encodes a 318 amino acid protein with a molecular mass of  31.7 kDa and a calculated pI of 4.53 and belongs to the Lip family that consists of 21 members in M. tuberculosis.Attemptsto express Rv1399c as a soluble protein using different E. coli bacterial strains, different temperatures, media and fusion protein constructs were unsuccessful. In all cases, the recombinant protein was expressed as inclusion bodies with the highest expression level obtained from the pDest 17O/I construct using the BL21 (DE3)pLysS cells grew in LB medium after 4 h of IPTG induction (Fig. 1). Purification and refolding of Rv1399c Given the large quantity of insoluble material obtained [up to 160 mg of protein from 6 L of cell culture (Table 1, Fig. 1 lane 4 )], we looked for optimal conditions to solubilize Rv1399c using a new r efolding analytical approach by sample dilution consisting of a rapid screening of different buffers in 96-well microtiter plates coupled to spectrophotometric analyses [12]. Among the 96 refolding conditions tested, 1 4 of t hem y ielded soluble Rv1399c based on the turbidity measurement at 340 nm. The choice of the final refolding buffer was guided by the optimum refolding yield. Rv1399c was refolded at 4 °Cover48husingthe buffer composition identified in the analytical s tep (50 m M Tris/HCl, pH 7.0). Ó FEBS 2004 Biochemical characterization of Rv1399c from M. tuberculosis (Eur. J. Biochem. 271) 3955 The annotation of Rv1399c as a putative LH prompted us to find a s uitable substrate, such a s t riglycerides and vinyl esters, known to be s electively hydrolyzed by a l arge number of NLH as well as LH. The preliminary biochemical experiments allowed us to follow the specific activity of Rv1399c along the refolding process a nd to estimate the refolding y ield (Table 1). Using tripropionin as a substrate, a specific activity of 80 UÆmg )1 was obtain ed a fter the dilution step and increased 6· after two days. After the concentration s tep ( 2–3 mgÆmL )1 ), the specific activity increased to 995 UÆmg )1 , suggesting that some insoluble material was s till refolded during t his process. The last freezing/thawing step appeared to be an efficient and powerful purification procedure. Indeed, whereas the total activity was not affected significantly, the specific activity increased from 1050 to 1350 UÆmg )1 . This was due to the presence of precipitated material accounting for 13% of the total protein and arising from unfolded or misfolded Rv1399c that could be easily eliminated by centrifugation. Finally, 80 mg of purified active Rv1399c, out of 160 mg present in inclusion bodies, were recovered after the Ni + -column purification step, resulting in a refolding yield of 50% with a purity of  98% based on SDS/PAGE gel (Fig. 1, lane 6). Moreover, DLS analysis indicates that Rv1399c behaves as a monodisperse in solution with an estimated hydrodynamic radius consistent with its molecu- lar size for a monomeric protein (data not shown). Biochemical characterizations of Rv1399c Catalytic properties. The specific activity determined against vinyl esters and triacylglycerols clearly shows that Rv1399c specifically hydrolyzes short- chain esters (Table 2). In all cases, the shape of the Michaelis–Menten represen- tation curves [V ¼ f([S]); V is the velocity (U) and [S] is the substrate concentration (m M )] is hyperbolic and the max- imal activity was measured in the soluble concentration range of the selected esters. Indeed, the K 1/2 value of Rv1399c using v inyl acetate (8 m M ) is similar to those determined for pig liver esterase and acetylcholinesterase (4 m M ). Similarly, the K 1/2 value of Rv1399c (2.76 m M ) using soluble t riacetin at a concentration far b elow its CMC (105 m M ) [24] is similar to that of pig liver esterase (4 m M ) but is lower to that of acetylcholinesterase (30 m M ). The kinetic behavior of Rv1399c using short-chain soluble vinyl esters and triacylglycerols along with the lack of d etectable activity using insoluble vinyl esters (vinyl laurate) or triacylglycerols (trioctanoin, olive oil) unambiguously clas- sify Rv1399c as a NLH rather than a LH. However, Rv1399c is able to hydrolyze a wide range of ester bonds and does not show a substrate specificity towards the alcohol or the acid moiety of short-chain esters (Table 2). The pH (Fig. 2A) and temperature (Fig. 2B) stability profiles of Rv1399c have been also investigated. The choice of tripropionin (9.3 m M ) as sub strate was g uided by t he high stability of this compound in the w ide range of temperature used. Our data indicate that Rv1399c is very sensitive to t he pH as no activity was recorded after 1 h incubation in acetate buffer (pH 4.0). The enzymatic activity of Rv1399c increases up to 4· at 45 °C and then dropped rapidly at higher temperatures with no detectable activity above 60 °C, indicating that Rv1399c cannot be considered as a thermophilic NLH. The catalytic triad From a kinetic point of view, we have established that Rv1399c is a NLH, consistent with the sequence homology detected between Rv1399c and other serine carboxyl ester Table 1. Flowsheet of the Rv1399c procedure purification and refolding. Tot al activity, 1 U ¼ 1 lmol of s ubstrate hydrolysed per min. Steps Protein amount (mg) Total activity (U) Amount of active protein (mg) Specific activity (UÆmg )1 ) % of refolding Inclusion bodies after washing 1040 – – – – Ni ± chelating Sepharose and concentration 160 – – – – Refolding by dilution (T0) 160 12880 9.5 80.5 5.9 Refolding by dilution (day two) 160 77760 57.6 486 35 Concentration and centrifugation 104 103480 76.7 995 48 Desalting column and concentration 92 92000 68.1 1000 42.6 After freezing 92 96600 71.5 1050 44.7 After thawing and centrifugation 80 108000 80 1350 50 1 116 kDa 66 kDa 45 kDa 35 kDa 25 kDa 234 5 6 7 Fig. 1. SDS/PAGE for expression and refolding of Rv1399c in E. coli. Protein samples were lo aded on a 14% S DS/PAGE under reducing and Coomassie-blue staining conditions. Lanes 1 and 7, molecular mass markers (Fermentas); lane 2, E. coli proteins ( 30 lg) before IPTG induction; lane 3, E. coli proteins ( 35 lg) after IPTG in duc- tion; lane 4, purified Rv1399c (14.5 lg) eluted from the Ni-nitrilotri- acetic acid column; lane 5, partially refolded Rv1 399c ( 6.8 lg); lane 6, refolded Rv1399c after the freeze/thaw step (11 lg). The apparent molecular mass of 36313 Da as estimated by Electrospray mass spectrometry is due to the p resence, at the N-terminus, o f t he His 6 -tag and the additional 21 residues from the expression plasmid. 3956 S. Canaan et al. (Eur. J. Biochem. 271) Ó FEBS 2004 hydrolases (Fig. 3). These enzymes share a functional catalytic triad made of a catalytic nucleophile serine, associated to a proton c arrier histidine and a c harge r elaying aspartic (or glutamic) acid. To further investigate the biochemical characterization of the enzyme, we have titrated these key residues that form the catalytic triad of Rv1399c. Catalytic serine. Diethyl p aranitrophenyl phosphate (E600), a specific powerful inhibitor of s erine hydrolases was assayed on Rv1399c. As shown in Fig. 2D, the purified enzyme was incubated with different ratio of E600. In each case, the kinetics of inactivation of Rv1399c have been monitored b y measuring the remaining activity, a s a function of time, using tripropionin as a substrate. Table 2. Comparison of the maximum specific activities of Rv1399c, acetylcholinesterase and pig liver esterase recorded using vinyl esters and triacylglycerols as substrates. All specific activities are in lmol o f acid released per min and per mg of enzyme in 2.5 m M Tris buffer pH 7.0 containing 100 m M NaCl at 25 °C. Substrate con centrations (indicatedinparentheses)areexpressedinm M ; ND, non determine d. Acetylcholine- sterase and pig liver esterase values are taken from Table 1 of referenc e [7]. Enzyme Substrate (concentration) Vinyl acetate (315) Vinyl propionate (80) Vinyl butyrate (20) Vinyl laurate – Triacetine (300) Tripropionin (9.28) Tributyrin (9.1) Trioctanoin – Olive oil – Acetylcholine (33) Rv1399c 610 1618 1123 0 184 1350 456 0 0 0 Acetylcholinesterase 970 210 0 0 450 0 0 0 0 1420 Pig liver esterase 320 300 470 0 60 50 70 0 0 ND 100 AB CD 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 43 567 7 6 5 4 3 2 1 0 8910 pH 432 56789 p H Residual activity (%) Residual activity (%) Activity (unit) Activity (%) 0 10203040506070 Temperature (C) WT Mutant Time (min) 0.5 15 43 1 for 10 1 for 2 pKaWT pKaMut Fig. 2. Kinetic assays of Rv1399c. (A) pH stability, (B) temperature dependence. (C) Titration curves of wt Rv1399c (s) and the Arg213Ala mutant (h) catalytic histidine residue. The curve profiles corresponding to pH values below 3.75 have been extrapolated due to Rv1399c instability at acidic pH. (D) Rv1399c inhibitory effect using the E600 inhibitor as function of time. The protein/inhibitor ratio is indicated. Enzyme a ctivity was determined as described in the Methods section. Ó FEBS 2004 Biochemical characterization of Rv1399c from M. tuberculosis (Eur. J. Biochem. 271) 3957 Rv1399c is strongly inhibited by E 600 with a K I value in the 7to30· 10 )10 M range, suggesting that the catalytic serine is highly reactive. Moreover, the inhibition by the E600 is not influenced by the presence of detergent, in contrast to lid-containing human gastric and pancreatic lipases, sug- gesting that the catalytic s erine o f R v1399c is fully accessible to the solvent. Catalytic histidine. It is widely assumed that serine carboxyl ester hydrolases, like serine proteases, require an appropriate protonation state of essential catalytic residues in the active p H range. The imidazole ring of the catalytic His must be in a neutral state to capture the hydrogen of the catalytic serine for an efficient nucleophilic attack of the substrate ester bond by the serine alcoholate. The shape of the titration curve of the catalytic histidine shows an apparent pKa value of the essential histidine estimated to 4.1 (Fig. 2C). This acidic pKa shift of the histidine residue has been described previously for carboxypeptidase Y [25] and gastric lipases [26–28] but no clear explanation of such an atypical property for t he enzyme function has b een yet established. 3D Model of Rv1399c Novel member of the HSL family. BLAS T searches against t he 163 currently available microbial genomes reveal that Rv1399c is only found in Mycobacteria species, e.g. Mycobacterium bovis AF2122/97 and Mycobacterium t uber- culosis CDC1551,withe-values in the 10 )150 range corres- ponding to 91% and 53% identity, respectively. Moreover, BLAST searches against the Protein Data Bank identified four top-ranked hits with sequence identity ranging from 44% to 31%. These structural homologs, which are members of the human HSL protein family, include the thermophilic esterase from Bacillus acidocaldarius [29] (44.2% of sequence identity), the thermophilic esterase from Archaeoglobus fulgidus [30] (43%), the brefeldin A esterase from B. subtilis [31] (33%) and the heroin esterase from Rhodococcus sp. strain H1 [32] (31%) (Fig. 3). Two invariant sequence motifs within members of the HSL protein family, namely the active site residues Ser162 and Asp260 and the consensus motif HGGG, are conserved in Rv1399c. In addition, a significant sequence identity (22%) can be found between Rv1399c and the catalytic domain of the HSL, indicating that Rv1399c is likely a new member of the HSL family [31], which to date, comprises more than 65 members. A model of Rv1399c was built with the SWISS MODEL server using, as templates, the co ordinates of structural homologues of the a/b hydrolase fold family [33] that present the highest sequence homologies (see above) with Rv1399c: serine hydrolase (accession code 1evq) [29], and carboxylester hydrolase (1jji) [30]. This m odel reveals the overall topological organization of Rv1399c, predicts the location of the catalytic triad, provides a valuable template for further structure-function studies and is consistent with our biochemical data. As expected, Rv1399c consists of three domains. The largest domain encompasses strands b1 to b6 a nd strand b7 to the C-terminus. The central b sheet is composed of 7 parallel b strands associated to an anti- parallel strand (b2) and is surrounded by 5 helices (a1, a2, a3, a7anda8). T he second domain c onsists of helices a4, a5 and a6 a ll clustered on the top of t he enzyme, as described by Wei et al. [31] (Fig. 4). The third domain, which consists of the 50 first amino acids, could not been modeled. This Fig. 3. Amino-acid sequence alignment between Rv1399c and four non lipolytic hydro- lases (NLH) of known 3D structure. The alignment was performed using the T - COFFEE and ESPRIT programs (available from the expasy web site). Conserved residues are boxed and those similar are indicated with a black background. Residues involved in the catalytic triad are indicated by w, while Arg213 (j) a long with the three hydr ophobic residues Trp92, Phe219 and Trp222, involved in the substrate s pecificity, are indicated by m. 3958 S. Canaan et al. (Eur. J. Biochem. 271) Ó FEBS 2004 α4 α5 α6 α7 α3 α8 β8 β7 β6 β5 β3 β4 β2 β1 α1 α2 Asp 260 His 290 Ser 162 Asp214 Asp214 Asp212 Asp212 Asp161 Asp161 Arg213 Arg213 Phe219 Phe219 Trp222 Trp222 Trp92 Trp92 Gly91 Gly91 Gly90 Gly90 Gly89 Gly89 Gly291 Gly291 T y r292 T y r292 Tyr190 Tyr190 Tyr190 Tyr190 His290 His290 His88 His88 Ser162 Ser162 Rv1399c Ag85c Fusarium solani pisi cutinase Bacillus subtilis lipase A A B C Fig. 4. Model of Rv1399c. (A) Ribbon diagram of Rv1399c model. The central b-sheet and surrounding a-helices are shown in green and red, respectively. Residues of the catalytic triad (Ser1 62, Asp260 and H is290) are shown in CPK and are colored in blue. (B) Close-up stereoview of the active s ite poc ket w ith b ound E 600 i nhibito r (red ) sh owing re sidue s of the oxyanion h ole. Residu es th at co uld play a role in substrate recognition are also indicated. (C) Molecular surface of Rv1399c model, Ag85C (1DQZ), Fusarium solani pisi cutinase (2CUT) and Bacillus subtilis lipase A (1ISP), viewed i n a sim ilar o rientation and looking down the active site, with s urface p atche s made f rom a lipha tic o r h ydrophobic residu es (Val, Ala, Leu, Ile, Trp, Tyr, Phe and Met) indicated in yellow. In all cases, the catalytic serine residue is shown in red. Ó FEBS 2004 Biochemical characterization of Rv1399c from M. tuberculosis (Eur. J. Biochem. 271) 3959 model is in agreement with our CD data showing two minima at 205 and 215 nm, indicating that Rv1399c is composed of 22% o f a-helices, 25% of b-strand a nd 39% o f random coil. The catalytic triad is located at the bottom of a small pocket, 20 A ˚ length and 10 A ˚ wide. The pocket is made by three distinct surface regions: a 10-residue loop connecting helix a8tostrandb8, a five-residues loop connecting strand b3 to helix a1 and two small h elices (a4anda6). The catalytic site consists of a functional catalytic triad found in all serine enzymes of the a/b hydrolase fold family in which Ser162 is the nucleophile residue, His290 is the proton carrier and Asp260 is the charge relay network. Ser162 is located within the Ônucleophilic elbowÕ connecting strand b5 and helix a3, while His290 and Asp260 e merge from the b8-a8andb7-a7 loops, respectively. Another important feature of the catalytic machinery is the so-called oxyanion hole necessary for the stabilization of the oxyan- ion transition state [34]. In Rv1399c, the oxyanion hole is likely to be formed by the amides of Ala163, Gly89 and Gly90, the latter’s being found in the HGGG consensus sequence motif characteristic of members of the HSL family [35]. Our model shows that the architecture of the active site could accommodate an E600 inhibitor molecule covalently bound to the catalytic Ser162 without d rastic conforma- tional changes (Fig. 4C). The acyl binding pocket (Fig. 4C) is delimited at one end by the three hydrophobic side chains Trp92, Phe219 and Trp222 that could play the role of filter thus preventing binding of substrate with an acyl chain larger than eight carbon atoms. This feature could explain the absence of Rv1399c activity against triacylglycerols or esters with longer carbon chain (Table 2). Moreover, t he three charged residues Asp212, Arg213, Asp161 located at the periphery of the pocket could have a role in substrate recognition, suggesting a preference for a polar noncationic substrate as a natural substrate since Rv1399c does not exhibit any catalytic activity against acetylcholine (Table 2). A structural s imilarity search, performed with DALI using the coordinates of t he Rv1399c model as a template identified numerous homologous proteins from the a/b hydrolase fold family ( Z score v alues >10), including Ag85C from M. t uberculo sis, a major protein component of the cell w all [4]. The rmsd value between these two structures is 3.2 A ˚ for 210 residues with only 11% of sequence identity. Although, Ag85C and Rv1399c share a similar fold organization, the architecture of their active sites differ markedly with that of Rv1399c (Fig. 4C). The mo lecular surface of the model does not reveal a large hydrophobic patch around the catalytic serine consis- tent with the lack o f a lipolytic activity (Fig. 4C). Indeed, t he high number of hydrophobic residues identified in the vicinity of the active site pocket of several lipases, such as cutinase [20,22] or B. subtilis lipase A [23], emphasizes the key role of this h ydrophobic surface patch for binding medium and long chain substrates, s uch as trioctanoin, vinyl laurate and olive oil as suggested by Fojan et al.[36]. Similarly, Ag85C possesses such hydrophobic determinants to effi ciently bind to myc olic acids [4]. In contrast, a reduced hydrophobic patch at the periphery of the active site pocket of Rv1399c is consistent with its with the lack of hydrolysis activity against insoluble substrates with medium or long acyl chain. This suggests that although M. tuberculosis Rv1399c and Ag85C share a similar fold, they have different substrate specificities and thus have nonrelated function. The Rv1399c model could explain the pK a shift of the catalytic histidine observed experimentally. Indeed, the presence of Arg213 along with the proximity of the charge relay Asp260 within the active site could be responsible for the modification of the catalytic properties of the catalytic histidine. The strength of the His290-Asp260 hydrogen bond could be reduced due to the proximity of the Arg213 guanidinium moiety that favor salt bridge formation with theAsp260sidechain( 5.5A ˚ ¢ ) and induces a charge repulsion with the His290 imidazolium (  2.5A ˚ ¢ ). This long range hydrogen bond distance observed between His290 and Asp260, along with the close proximity of His290 to hydrophobic residues from the b8-a8 loop (Trp189, Tyr190), could f avor the displacement of the equilibrium toward the neutral state of His290. To attest this hypothesis, an Arg213Ala mutant has been expressed and characterized. The specific activity of the Ala213 mutant, using tripropionin as substrate, remains identical (1280 UÆmg )1 )tothewt enzyme (1350 UÆmg )1 ) but the apparent pKa increases to  5.5. However, the titration curve shows a plateau at acidic pH indicating that Arg213 is not the only residue that dictates the a cidic catalytic profile of His290 (Fig. 2C). Concluding remarks In summary, we have expressed, purified and refolded the M. tu berculosis Rv1399c protein from inclusion bodies with a h igh r efolding yield (50%) and have biochemically defined Rv1399c a s a non lipolytic hydrolase. This enzym e efficiently hydrolyzes short-chain synthetic substrates such as triacylglycerols and vinyl esters and the three-dimensional model is fully consistent with our experime ntal data. We thus propose to rename Rv1399c as NLH-H instead of LipH. The ability to express and refold Rv1399c in large amounts in E. coli represents a key step for f urther crystallization experiments aimed at solving its three- dimensional s tructure. Although the nature of Rv1399c substrate and the role of this enzyme in the M. tuberculosis life cycle remain to be investigated, our study represents a key s tep towards the e lucidation of the biological function of Rv1399c. Given the broad substrate specificity of Rv1399c, this enzyme may participate in the detoxification pathway of the intracellular lipid metabolism. Recent findings about of LipF, another member of the Lip family, to belong to a gene cluster related to virulence [37], emphasize future studies aimed at understanding the detailed bioch emical characterization combined to a crys- tallographic approach of members of the Lip family. Acknowledgements We than k Nadine Honore ´ for p roviding us with the M. tuberculosis cosmids and BACs libra ries. We are gr ateful to Rob ert Verger, Louis Sarda, Steward Cole and Mary Jackson for fruitful discussions, an d Christophe Bignon and Renaud Vincentelli for their tec hnical assist- ance. This work was supported by a grant from the 5th PCRDT program of the European Union (acronym X -TB) and by the French national network of Genopole. 3960 S. Canaan et al. (Eur. J. Biochem. 271) Ó FEBS 2004 References 1. Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S.V., Eiglmeier, K., Gas, S., Barry 3rd, C.E., et al. 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