Expression and characterization of the protein Rv1399cA novel carboxyl esterase structurally related to the HSL family Ste´phane Canaan1, Damien Maurin1, Henri Chahinian2, Be´ne´dicte Po
Trang 1Expression and characterization of the protein Rv1399c
A novel carboxyl esterase structurally related to the HSL family
Ste´phane Canaan1, Damien Maurin1, Henri Chahinian2, Be´ne´dicte Pouilly1, Ce´cile Durousseau1,
Fre´de´ric Frassinetti1, Lore´na Scappuccini-Calvo1, Christian Cambillau1and Yves Bourne1
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 to the Lip family, including various
nonlipolytic and lipolytic hydrolases Driven by a structural
genomic approach, we have biochemically characterized the
Rv1399c gene product, LipH, previously annotated as a
putative lipase Rv1399c was overexpressed in E coli as
inclusion bodies and refolded 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 paranitrophenyl phosphate
(E600) These kinetic behaviors unambiguously classify
Rv1399c as a nonlipolytic rather than a lipolytic hydrolase
Sequence alignment reveals that this enzyme belongs to the
a/b hydrolase fold family and shares 30–40% amino acid
sequence identity with 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 of a solvent accessible pocket lined by neutral or charged residues Based on this model, kinetic data of the Arg213Ala mutant partially explain the role of the guanid-inium moiety, located close to His290, to confer an unusual low pH shift of the catalytic histidine in the wild type enzyme Overall, these data identify Rv1399c as a new nonlipolytic hydrolase from M tuberculosis and we thus propose to reannotate its gene product as NLH-H Keywords: esterase; nonlipolytic hydrolase; Rv1399c; tuber-culosis
The recent elucidation of the complete sequence of
Myco-bacterium tuberculosisgenome [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 the 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 ester hydrolases, 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 tuberculosis MPT51 protein (FbpC1) [5], which is involved in 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 and become markedly 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 distinction between lipases and esterases was estab-lished recently from the comparison of the K1/2 values of these two classes of carboxyl ester hydrolases using partially soluble triacylglycerols and vinyl esters as substrates [7] Where a lipase can also be considered an esterase, the K1/2 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 the family denote nonlipolytic hydro-lases (NLH) Therefore, without a detailed biochemical
Macro-mole´cules Biologiques, AFMB UMR 6098, CNRS, 31 Chemin Joseph
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, hormone-sensitive lipase; NLH, nonlipolytic
hydrolase; LH, lipolytic hydrolase; IB, inclusion bodies; IPTG,
E600, diethyl para-nitrophenyl phosphate; DLS, dynamic light
scattering.
(Received 7 June 2004, accepted 17 August 2004)
Trang 2characterization, the Lip term could be confused to refer to
a NLH, and the original accession number should be used
to avoid confusion
Here we report the cloning, expression and refolding of
the M tuberculosis Rv1399c gene product, a member of 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 triacylglycerols 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 for 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 Ni+-agarose gel was obtained from Amersham
Biosciences Vinyl acetate, vinyl propionate, vinyl butyrate,
triacetin, tributyrin 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¢- and 3¢-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 the 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
thio-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 promoter To overcome this problem, a derivative
of the pDest 17 plasmid, pDest 17O/I, was constructed from
the pET-Dest42 plasmid and contains a
PshAI-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 binding to its specific DNA binding site on Lac O
Mutagenesis Site-directed mutagenegis, which was per-formed using the Quickchange site-directed mutagenesis system (Stratagen, La Jolla, CA, USA), was used for the mutation of Arg213fi Ala The oligonucleotides used 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 codharbor-ing region Cells were grown at 37C
in LB medium containing 100 lgÆmL)1 ampicillin and
34 lgÆmL)1chloramphenicol until a D600value between 0.6 and 0.8 was reached Protein expression was induced by adding 2 mMIPTG for 4 h at 37C and then 16 h at 15 C Rv1399c was highly overexpressed but found exclusively in inclusion bodies
Purification of inclusion bodies Cells were harvested at
4C by centrifugation at 9000 g for 15 min The pellet was resuspended in ice-cold lysis buffer [50 mM Tris/HCl,
pH 8.0, 150 mM NaCl, 10 mM imidazole, 1 mM EDTA, 0.1% (v/v) Triton X-100, 0.25 mgÆmL)1of lysozyme and
1 mM phenylmethanesulfonyl fluoride] and stored at )80 C overnight The pellet was thawed on ice for 1 h and then 10 lgÆmL)1 DNase and 20 mM MgSO4 (final concentration) were added to the cell suspension for 30 min Cells were disrupted by ultrasonication (10· with a 15 s cycle) using a Branson Sonifier 450 Inclusion bodies were separated from the cell extract by centrifugation at 17 000 g for 30 min The pellet was then washed with 10 mMTris/ HCl, pH 8.0 and 150 mMNaCl, 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 4C overnight in a
40 mL solution containing 10 mM Tris/HCl, pH 8.0,
150 mM NaCl and 6M guanidine hydrochloride The solubilized protein was clarified from insoluble material at
4C by centrifugation at 17 000 g for 15 min The super-natant was loaded (3 mLÆmin)1) onto a Ni+-agarose column (1 mL resin for 5 mg of recombinant protein) previously equilibrated with buffer A (10 mM Tris/HCl,
pH 8.0, 150 mM NaCl, 10 mM imidazole and 8M urea) The column was then washed using five column volumes of 5% and 10% of buffer B (buffer A + 500 mMimidazole) Enzyme was eluted with 50% of buffer B; fractions of eluted peaks containing purified Rv1399c were analyzed by SDS/ PAGE [11], pooled and concentrated up to 4–5 mgÆmL)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 conditions were determined by a refolding method
Trang 3based on the measurement of the turbidity at 340 nm using
a 96-well plate [12] The final refolding conditions consisted
of diluting Rv1399c 20· in 50 mMTris buffer, pH 7, at 4C
for 2 days Rv1399c was concentrated up to 2–3 mgÆmL)1
and traces of urea and imidazole were removed using a
desalting column (HiPrepTM26/10, Amersham Biosciences)
Rv1399c was then concentrated up to 3 mgÆmL)1 and
stored overnight at)20 C, and after thawing, the active
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 4C and can be stored at
)20 C for several months Protein concentration was
deter-mined by measuring at A280 using e280¼ 1.399 mg)1Æ
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 programCDNN(CD
SPEC-TRA DECONVOLUTION, version 2.1) Rv1399c (0.2 mgÆmL)1)
spectra were recorded at 20C between 185 and 260 nm in
10 mMTris/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 20C with filtered (Millex syringe filters, 0.22 lm;
Millipore Corp.) protein samples (12 lL at 3 mgÆmL)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 25C using a pH-stat (TTT 80, Radiometer,
Copenha-gen, Denmark) for at least 5 min Assays were performed in
30 mL of 2.5 mM of Tris/HCl buffer, pH 7.0 and 0.1M
NaCl Release of fatty acid was titrated with 0.1MNaOH
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 lauric 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 mM NaCl and: 150 mM sodium acetate,
pH 4.0; 150 mM sodium acetate, pH 5.0; 150 mM Mes,
pH 6.0; 150 m Tris/HCl, pH 7.0; 150 m Tris/HCl,
pH 8.0 and 150 mMglycine, pH 9.0 The residual activity was determined potentiometrically at 25C at pH 7.0 using tripropionin at the concentration of 9.3 mMas 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 curve was performed using vinyl butyrate (20.7 mM) 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 25C with different E600 inhibitor/enzyme molar ratio (2 and 10) in a final volume 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 thePROCHECKprogram [17] Sequence align-ment was performed initially using the multiple sequence alignment softwareT-COFFEE[18], displayed withESPRIT[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 Attempts to 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 refolding 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, 14 of them yielded 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 4C over 48 h using the buffer composition identified in the analytical step (50 mM Tris/HCl, pH 7.0)
Trang 4The annotation of Rv1399c as a putative LH prompted
us to find a suitable substrate, such as triglycerides and vinyl
esters, known to be selectively hydrolyzed by a large number
of NLH as well as LH The preliminary biochemical
experiments allowed us to follow the specific activity of
Rv1399c along the refolding process and to estimate the
refolding yield (Table 1) Using tripropionin as a substrate,
a specific activity of 80 UÆmg)1 was obtained after the
dilution step and increased 6· after two days After the
concentration step (2–3 mgÆmL)1), the specific activity
increased to 995 UÆmg)1, suggesting that some insoluble
material was still refolded during this 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 [V¼ f([S]); V is the velocity (U) and [S] is the substrate concentration (mM)] is hyperbolic and the max-imal activity was measured in the soluble concentration range of the selected esters Indeed, the K1/2 value of Rv1399c using vinyl acetate (8 mM) is similar to those determined for pig liver esterase and acetylcholinesterase (4 mM) Similarly, the K1/2 value of Rv1399c (2.76 mM) using soluble triacetin at a concentration far below its CMC (105 mM) [24] is similar to that of pig liver esterase (4 mM) but is lower to that of acetylcholinesterase (30 mM) The kinetic behavior of Rv1399c using short-chain soluble vinyl esters and triacylglycerols along with the lack of detectable 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 mM) as substrate was guided by the high stability of this compound in the wide range of temperature used Our data indicate that Rv1399c is very sensitive to the
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
60C, 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
Steps
Protein amount (mg)
Total activity (U)
Amount of active protein (mg)
Specific
1
116 kDa
66 kDa
45 kDa
35 kDa
25 kDa
Fig 1 SDS/PAGE for expression and refolding of Rv1399c in E coli.
Protein samples were loaded on a 14% SDS/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
induc-tion; lane 4, purified Rv1399c (14.5 lg) eluted from the
Ni-nitrilotri-acetic acid column; lane 5, partially refolded Rv1399c (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 presence, at the N-terminus, of the His6-tag
and the additional 21 residues from the expression plasmid.
Trang 5hydrolases (Fig 3) These enzymes share a functional
catalytic triad made of a catalytic nucleophile serine,
associated to a proton carrier histidine and a charge relaying
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 paranitrophenyl phosphate (E600), a specific powerful inhibitor of serine 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 by measuring the remaining activity, as 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
Acetylcholine-sterase and pig liver eAcetylcholine-sterase values are taken from Table 1 of reference [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)
100
80
60
40
20
0
100
80
60
40
20
0
100 80 60 40 20 0
4
7 6 5 4 3 2 1 0
pH
4 3
pH
Temperature (C)
WT Mutant
Time (min)
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 activity was determined as described in the Methods section.
Trang 6Rv1399c is strongly inhibited by E600 with a KIvalue in the
7 to 30· 10)10Mrange, 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 serine of Rv1399c 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 pH 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 the enzyme function has been yet
established
3D Model of Rv1399c
Novel member of the HSL family BLAST searches
against the 163 currently available microbial genomes reveal
that Rv1399c is only found in Mycobacteria species, e.g
Mycobacterium bovisAF2122/97 and Mycobacterium
tuber-culosis CDC1551, with e-values in the 10)150range
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 coordinates 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 model 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 and 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, a7 and a8) The second domain consists of helices a4, a5 and a6 all clustered on the top of the 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
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) along with the three hydrophobic residues Trp92, Phe219 and Trp222, involved
in the substrate specificity, are indicated by m.
Trang 7α7
α3 α8
β8
β7
β6
β5 β3 β4 β2 β1 α1
α2
Asp 260
His 290 Ser 162
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 (Ser162, Asp260 and His290) are shown in CPK and are colored in blue (B) Close-up stereoview of the active site pocket with bound E600 inhibitor (red) showing residues of the oxyanion hole Residues that could 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 in a similar orientation and looking down the active site, with surface patches made from aliphatic or hydrophobic residues (Val, Ala, Leu, Ile, Trp, Tyr, Phe and Met) indicated in yellow In all cases, the catalytic serine residue is shown in red.
Trang 8model is in agreement with our CD data showing two
minima at 205 and 215 nm, indicating that Rv1399c is
composed of 22% of a-helices, 25% of b-strand and 39% of
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 a8 to strand b8, a five-residues loop connecting strand
b3 to helix a1 and two small helices (a4 and a6)
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 nucleophilic elbow connecting
strand b5 and helix a3, while His290 and Asp260 emerge
from the b8-a8 and b7-a7 loops, respectively Another
important feature of the catalytic machinery is the so-called
oxyanion hole necessary for the stabilization of the
oxyan-ion transitoxyan-ion state [34] In Rv1399c, the oxyanoxyan-ion 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 drastic
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, the 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 similarity search, performed with DALI
using the coordinates of the Rv1399c model as a template
identified numerous homologous proteins from the a/b
hydrolase fold family (Z score values >10), including
Ag85C from M tuberculosis, a major protein component
of the cell wall [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 molecular surface of the model does not reveal a
large hydrophobic patch around the catalytic serine
consis-tent with the lack of a lipolytic activity (Fig 4C) Indeed, the
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 hydrophobic surface patch for binding
medium and long chain substrates, such as trioctanoin,
vinyl laurate and olive oil as suggested by Fojan et al [36]
Similarly, Ag85C possesses such hydrophobic determinants
to efficiently bind to mycolic 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 pKashift 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 the Asp260 side chain ( 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 favor 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) to the wt 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 acidic catalytic profile of His290 (Fig 2C)
Concluding remarks
In summary, we have expressed, purified and refolded the
M tuberculosisRv1399c protein from inclusion bodies with
a high refolding yield (50%) and have biochemically defined Rv1399c as a non lipolytic hydrolase This enzyme efficiently hydrolyzes short-chain synthetic substrates such
as triacylglycerols and vinyl esters and the three-dimensional model is fully consistent with our experimental 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 further crystallization experiments aimed at solving its three-dimensional structure 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 step towards the elucidation 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 biochemical characterization combined to a crys-tallographic approach of members of the Lip family
Acknowledgements
We thank Nadine Honore´ for providing us with the M tuberculosis cosmids and BACs libraries We are grateful to Robert Verger, Louis Sarda, Steward Cole and Mary Jackson for fruitful discussions, and Christophe Bignon and Renaud Vincentelli for their technical 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.
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