The 3D model of the lipase/acyltransferase from Candida parapsilosis, a tool for the elucidation of structural determinants in CAL-A lipase superfamily

Article abstract of DOI:10.1016/j.bbapap.2015.06.012

Abstract Because lipids are hydrophobic, the development of efficient bioconversions in aqueous media free of organic solvents is particularly challenging for green oleochemistry. Within this aim, enzymes exhibiting various abilities to catalyze acyltransfer reaction in water/lipid systems have been identified. Among these, CpLIP2 from Candida parapsilosis has been characterized as a lipase/acyltransferase, able to catalyze acyltransfer reactions preferentially to hydrolysis in the presence of particularly low acyl acceptor concentration and high thermodynamic activity of water (a_w > 0.9). Lipase/acyltransferases are thus of great interest, being able to produce new esters at concentrations above the thermodynamic equilibrium of hydrolysis/esterification with limited to no release of free fatty acids. Here, we present a 3D model of CpLIP2 based on homologies with crystallographic structures of Pseudozyma antarctica lipase A. Indeed, the two enzymes have 31% of identity in their primary sequence, yielding a same general structure, but different catalytic properties. The quality of the calculated CpLIP2 model was confirmed by several methods. Limited proteolysis confirmed the location of some loops at the surface of the protein 3D model. Directed mutagenesis also supported the structural model constructed on CAL-A template: the functional properties of various mutants were consistent with their structure-based putative involvement in the oxyanion hole, substrate specificity, acyltransfer or hydrolysis catalysis and structural stability. The CpLIP2 3D model, in comparison with CAL-A 3D structure, brings insights for the elucidation and improvement of the structural determinants involved in the exceptional acyltransferase properties of this promising biocatalyst and of homologous enzymes of the same family.

Full text of DOI:10.1016/j.bbapap.2015.06.012

Biochimica et Biophysica Acta 1854 (2015) 1400–1411
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Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbapap
The 3D model of the lipase/acyltransferase from Candida parapsilosis, a
tool for the elucidation of structural determinants in CAL-A
lipase superfamily
a
b
c
Maeva Subileau a, , Anne-Hélène Jan , Hervé Nozac'h , Marina Pérez-Gordo ,
⁎
Véronique Perrier a, Eric Dubreucq a
a
Montpellier SupAgro, UMR 1208 IATE, 2 Place Viala, F-34060 Montpellier cedex, France
iBiTec-S, Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), Commissariat à l'énergie atomique et aux énergies alternatives (CEA) Saclay, France
IIS-Fundación Jiménez Díaz, Department of Allergy Avda. Reyes Católicos, 2, Madrid, ES 28040, Spain
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 May 2015
Received in revised form 19 June 2015
Accepted 24 June 2015
Available online 27 June 2015
Because lipids are hydrophobic, the development of efficient bioconversions in aqueous media free of organic sol-
vents is particularly challenging for green oleochemistry. Within this aim, enzymes exhibiting various abilities to cat-
alyze acyltransfer reaction in water/lipid systems have been identified. Among these, CpLIP2 from Candida
parapsilosis has been characterized as a lipase/acyltransferase, able to catalyze acyltransfer reactions preferentially
to hydrolysis in the presence of particularly low acyl acceptor concentration and high thermodynamic activity of
water (aw N 0.9). Lipase/acyltransferases are thus of great interest, being able to produce new esters at concentra-
tions above the thermodynamic equilibrium of hydrolysis/esterification with limited to no release of free fatty
acids. Here, we present a 3D model of CpLIP2 based on homologies with crystallographic structures of Pseudozyma
antarctica lipase A. Indeed, the two enzymes have 31% of identity in their primary sequence, yielding a same general
structure, but different catalytic properties. The quality of the calculated CpLIP2 model was confirmed by several
methods. Limited proteolysis confirmed the location of some loops at the surface of the protein 3D model. Directed
mutagenesis also supported the structural model constructed on CAL-A template: the functional properties of var-
ious mutants were consistent with their structure-based putative involvement in the oxyanion hole, substrate spec-
ificity, acyltransfer or hydrolysis catalysis and structural stability. The CpLIP2 3D model, in comparison with CAL-A
3D structure, brings insights for the elucidation and improvement of the structural determinants involved in the ex-
ceptional acyltransferase properties of this promising biocatalyst and of homologous enzymes of the same family.
© 2015 Elsevier B.V. All rights reserved.
Keywords:
Biocatalysis
Protein engineering
Lipase/acyltransferase
CpLIP2
CAL-A
Green chemistry
1. Introduction
for example with lipases from Candida deformans, Mucor miehei, Rhizopus
delemar, Pseudomonas fluorescens, Rhizopus arrhizus or with lipase A from
Lipases (E.C.3.1.1.3.) are largely used biocatalysts in organic synthesis
[1–7], but their use in the oleochemical industry for the production of
fatty esters mostly relies on non-aqueous processes using hydrophobic
solvents [8]. Even if lipases offer the well-known advantages of enzymatic
processes, the development of greener processes is the next challenge for
the improvement of biotechnological transformations of oils and fats. Re-
actions in two-phase systems with water as the polar solvent and lipid as
the organic phase present the advantage of using no organic solvent and
allowing the solubilization of both enzyme and polar nucleophiles, and
lipids, in systems that mimic the natural form for lipase catalysis. Two
kinds of ester synthesis reactions can be distinguished. One is the esterifi-
cation of free fatty acids, where a lipase is used to catalyze reverse hydro-
lysis to reach the thermodynamic equilibrium of the hydrolysis/
esterification reaction. This has been successfully used in various studies,
P. antarctica [9–15]. With such lipases, a so-called transesterification can
also be obtained through the hydrolysis of an ester substrate followed
by the re-esterification of the released fatty acids with an alcohol acceptor.
A direct alcoholysis of an ester substrate can also be catalyzed by classical
lipases but this is not the main reaction in aqueous conditions with high
aw [9,14]. However, it has been shown that some lipases, described as li-
pases/acyltransferases, are able to catalyze alcoholysis preferentially to
hydrolysis even in the presence of low alcohol concentration and high
aw (N0.9). Contrarily to pure acyltransferases, that require highly activat-
ed substrates such as acyl-CoA [16], lipases/acyltransferases are able to
catalyze the transfer of acyl groups in aqueous media using simple esters
as substrates, such as triacylglycerols. Such enzymes and their applica-
tions have been described for several years by our group. The first one
was the CpLIP2 lipase/acyltransferase from Candida parapsilosis [17–21],
followed by CaLIP4 from Candida albicans [22] and CtroL4 from Candida
tropicalis [23]. These lipases/acyltransferases have been shown to
be homologous to C. albicans lipase 1 and CAL-A, the lipase A of
⁎
Corresponding author.
E-mail address: maeva.subileau@supagro.fr (M. Subileau).
http://dx.doi.org/10.1016/j.bbapap.2015.06.012
1570-9639/© 2015 Elsevier B.V. All rights reserved.

M. Subileau et al. / Biochimica et Biophysica Acta 1854 (2015) 1400–1411
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P. antarctica (formerly designated as Candida antarctica) [20,24] and to
belong to the P. antarctica lipase A-like α/β hydrolase superfamily
(abH38) [23,25,26] of the Lipase Engineering Database [27]. This super-
family has a very low degree of sequence identity with other classical li-
pases and thus constitutes an original family of fungal lipases [25,26]. It
is to be noted that CAL-A itself has a low acyltransferase character com-
pared to CpLIP2, and behaves more like classical lipases on this point (in
high water content media, CAL-A does not catalyze the production of
new esters at concentrations above the thermodynamic equilibrium of
hydrolysis/esterification [23]). Apart from those fungal lipases and an en-
zyme from Mycobacterium smegmatis reported to be able to catalyze
alcoholysis in aqueous conditions [28], the great potential of lipases/
acyltransferases for the development of environmentally friendly enzy-
matic processes has been poorly discussed in lipase literature. Compara-
ble “kinetically controlled” reactions have however been described for
acyltransfer catalysis in water abundant media with other enzymes,
such as for the synthesis of peptide bonds using proteases or for amine-
acylation catalyzed by penicillin-acylases [29–40]. Such biocatalysts also
display intrinsic properties that determine a kinetically controlled synthe-
sis with a superior transfer ratio compared to the corresponding “classi-
cal” hydrolases, which catalyze “equilibrium controlled” synthesis.
Likewise, the development of efficient fatty ester synthesis with enzymes
in biphasic aqueous media is an attractive response to green chemistry
challenges, not only for the advantages of the biocatalysis process itself
but also by facilitating the downstream processing (simple oil/water
phase separation). Within the few lipases able to catalyze alcoholysis
preferentially to hydrolysis in aqueous media, the lipase/acyltransferase
CpLIP2 from C. parapsilosis appears as the best candidate today for the de-
velopment of lipid bioconversions and functionalizations without a strict
control of aw [14,19,23] and has been described as of high industrial inter-
est [41–44]. With CpLIP2, competition of a short chain alcohol with water
has been shown to be highly favorable to the alcohol, even with a high aw
and a low thermodynamic activity of the alcohol [9,14,18,21,23].
Transesterification with this enzyme appears thus kinetically controlled
(very low rate of hydrolysis versus high rate of alcoholysis), which allows
the production of new esters at concentrations above the thermodynamic
equilibrium of hydrolysis/esterification. Other nucleophiles such as
amines or hydrogen peroxide have also been shown to be preferred nu-
cleophiles for this enzyme [45–47]. CpLIP2 has also been studied in biore-
actors in free or immobilized forms for fat and oil transesterification and
interesterification in aqueous or solvent-free systems [48–50]. Another
interesting feature of CpLIP2 and its homologs CtroL4 and CaLIP4 is their
high specific activity at low temperatures, with for example CpLIP2
displaying at 5 °C 65% of its alcoholysis activity at 30 °C, allowing their
classification as cold-active lipases contrary to their more distant homolog
CAL-A (31% identity with CpLIP2) [51]. Within the CAL-A family, lipases
thus exhibit differences in their catalytic properties such as acyltransfer
ability, substrate specificity, thermostability and activity at low tempera-
ture [23,51,52].
The elucidation of the 3D structure of CpLIP2 is now essential to un-
derstand the structural determinants responsible for its exceptional cat-
alytic specificities, and to further improve them. Unfortunately, despite
numerous attempts, no crystal of CpLIP2 has been obtained yet and
crystallization experiments with CpLIP2 and several mutants and ho-
mologs with high acyltransferase activity are still under way. We have
therefore built a 3D model based on its homology with CAL-A. This
structural model has then been cross-validated by the study of several
mutants obtained by site-directed mutagenesis, limited proteolysis ex-
periments and molecular docking.
2. Materials and methods
2.1. Modeling
CAL-A 3D structures (PDB ID: 2VEO and PDB ID: 3GUU) are the only
currently available structural templates with sequence identity versus
CpLIP2 above 15%. MUSTER [57], I-TASSER [58], and ROBETTA [59–61]
servers were used for the construction and refinement of CpLIP2 3D
models. Evaluation of the quality of the obtained 3D structures was per-
formed with Verify3D [62,63] and ERRAT [64] servers. Verify 3D gives a
numerical value for each residue which is an estimation of the 1D/3D
compatibility considering the relevance of the residue positioning in
its structural environment (polarity, accessibility). Residues with high
positive scores are considered to have consistent positioning. ERRAT
gives (i) “error values” for each 9-residue sliding window based on sta-
tistics of atomic contacts: the smaller the values, the better the folding,
and (ii) an “overall quality factor” which is empirically correlated with
the global correctness of the 3D structure. PyMOL [65] and UCSF Chime-
ra molecular graphic systems [66] were used for visualization of the 3D
structures. Molecular docking within the CpLIP2 model structure was
performed with AutoDock Vina [67], via USCF Chimera.
2.2. Plasmids, strains, enzymes and reagents
For the production of recombinant CpLIP2 and CAL-A, transformed
strains of Pichia pastoris previously produced by our group were used
[23,41]. For the production of CpLIP2 mutant enzymes, competent
cells of Escherichia coli XL1-Blue MRF' (Stratagene, La Jolla, CA, USA)
were used for DNA propagation. P. pastoris GS115, pPIC9K vector, Ge-
neticin and ampicillin were from Invitrogen (Life Technologies SAS,
Saint Aubin, France). SacI restriction enzyme and RNaseA were pur-
chased from Roche Diagnostics AG (Rotkreuz, Switzerland). All lipase
substrates and reagents were purchased from Sigma-Aldrich (Lyon,
France) and were of analytical grade.
2.3. Sequence analysis
Sequence visualization and multisequence alignments were generat-
ed using Seaview software [68] and Clustal [69] software. Protein families
were organized on the level of homologous families and superfamilies
based on their sequence similarity. BLAST [53] searches were performed
against the current version of the non-redundant sequence database of
NCBI [70] or of the Lipase Engineering Database [27].
The high potential of CpLIP2 for industrial applications has
prompted us to further study the structure and function of this enzyme
to better understand its peculiar catalytic features. CpLIP2 is a 465-
amino acid polypeptide including a 16-residue N-terminal signal
peptide in its native form [20]. If a BLAST alignment [53] with CpLIP2 se-
quence currently gives more than 500 protein sequences with more
than 25% identities and 40% positives, most of them are putative or hy-
pothetical proteins obtained by systematic genome sequencing without
further annotation regarding their biochemical and enzymatic charac-
teristics. The closest sequence to CpLIP2 with elucidated 3D structure
corresponds to that of CAL-A (PDB ID: 2VEO and PDB ID: 3GUU) [54].
The 3D structure of CAL-A itself does not exhibit much structural homol-
ogies with other known lipases, except some similarity with the lipases
of Candida rugosa, described as lipases having a narrow tunnel to ac-
commodate the acyl group but a wide alcohol binding site [2,54,55]
and a lid-like peptide [25] that can play a role in interfacial activation
[56].
2.4. Limited proteolysis
Four proteases were selected for the limited proteolysis of CpLIP2.
Trypsin and chymotrypsin were used for their high specificity (respec-
tively 36 and 47 potential recognition sites in CpLIP2) while pepsin
(99 potential sites) and papain were chosen for their lower specificity.
The optimized proteolysis conditions with the four proteases were 10
and 20 min reaction time with trypsin, chymotrypsin and papain and
20 and 60 min with pepsin. Proteolysis was performed at 35 °C with a
1:100 protein:protease molar ratio. Analysis of the fragments on SDS-
PAGE allowed the selection of 18 bands that were cut out of the gel

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M. Subileau et al. / Biochimica et Biophysica Acta 1854 (2015) 1400–1411
and further digested for Maldi-TOF analysis (BiFlex III Maldi-TOF mass
spectrometer, Bruker Daltonik, Bremen, Germany) [71] using the
dried-droplet spot method, at the Functional Proteomics Laboratory of
Montpellier (INRA). Reflector spectra were obtained over a mass
range of 600–3500 Da in the short pulse ion extraction mode using an
accelerating voltage of 19 kV. Spectra from 100 to 200 laser shots
were summed to generate a peptide mass fingerprint for each protein
digest. Two peptide ions generated by the autolysis of trypsin were
used as internal standards to calibrate the mass spectra. The automatic
monoisotopic mass was assigned using Bruker's SNAP™ procedure.
Only the 8 bands with the highest molecular weight (N20 kDa) gave re-
liable results, while the intensity of the signal for the 10 others was too
low. Fragment identifications through the Mascot database search (Ma-
trix Science, London) [72] were confirmed by correlation between the
molecular weight of the peptides obtained by Maldi-TOF analysis and
by the SDS-PAGE electrophoresis gel. Data from these two methods
were 2 kDa concomitant.
2.6. Transformation of P. pastoris and selection of clones for mutant
enzyme expression
The plasmids containing the CpLIP2 sequence modified by site di-
rected mutagenesis were used for the transformation of P. pastoris
GS115. Fifty micrograms of each lipase expression vector was linearized
with 80 U of SacI for 6 h at 37 °C. DNA was then purified and concentrat-
ed using Illustra GFX PCR DNA and Gel Band Purification kit (GE
Healthcare, Velizy, France) according to the reference protocol of the
manufacturer. Five micrograms of linearized and purified plasmids
was used to transform 80 μL of competent cells of P. pastoris GS115 by
electroporation according to the Invitrogen manual. After transforma-
tion, cells were grown and selected on RDB (Regeneration Dextrose
Base) plates (see the Pichia Expression System manual) for selection
of His+ transformants. Colonies were picked and cultured successively
3 times for 24 h at 28 °C in 250 μL of YPD medium (10 g·L−1 yeast ex-
tract, 20 g·L−1 peptone, 20 g·L−1 glucose) buffered with 50 mM potas-
sium phosphate pH 6.5. Five microliters of culture medium was then
dropped on YPD plates containing increasing concentrations of Genet-
icin (0.25, 0.75 and 2 g·L−1), as well as on YNB-Rhodamine B plates
(13.4 g·L−1 yeast nitrogen base w/o amino acid, 5 g·L−1 rapeseed oil,
0.5% (v/v) methanol, 10 mg·L−1 Rhodamine B, 0.04 mg·L−1 D-biotin,
20 g·L−1 agar, 50 mM potassium phosphate pH 6.5). Cultivations
were performed at 28 °C for 2 to 5 days. Clones for each lipase were se-
lected based on the size of the fluorescent halo on YNB-Rhodamine B
plates upon UV illumination and on their degree of resistance to Genet-
icin, related to the number of integrated DNA copies [76]. Colonies of se-
lected clones were picked and spread on YNB-Rhodamine B plates [77]
for strain subcloning. Selected clones were then grown 24 h at 28 °C in
YPD medium and stored at −80 °C in 200 g·L−1 glycerol for conserva-
tion and further use. The transformants were then cultivated for heter-
ologous lipase/acyltransferase production as described by Brunel et al.
[41].
2.5. Expression vector production for the mutants of CpLIP2
Mutant protein sequences were designed on Seaview software. Mu-
tated amino acids are listed in Table 1 (the amino-acid numbering of
CpLIP2 sequence differs from that used in earlier literature: in the work
presented here, the 16 amino acids of the native signal peptide were re-
moved). Site directed mutagenesis was performed by GeneArt (Life
Technology) on the plasmid containing the gene of CpLIP2 (pPIC9K)
and constructed by Brunel et al. [41]. The mutated gene was in frame
with the α-factor secretion signal from Saccharomyces cerevisiae, and
under the control of the AOX1 promoter. The resulting plasmids were
then propagated in E. coli XL1-Blue MRF' using thermal shock transfor-
mation [73]. Transformants were selected on LB plates (10 g·L−1
tryptone, 5 g·L−1 yeast extract, 10 g·L−1 NaCl and 15 g·L−1 agar) con-
taining 100 mg·L−1 ampicillin. Positive transformants were grown for
16 h in 2 mL of LB medium containing 100 mg·L−1 ampicillin and the re-
combinant plasmids were isolated using the rapid alkaline extraction
procedure described by Birnboim and Doly [74] and adapted by
Sambrook et al. [75].
2.7. Heterologous production in bioreactor
Recombinant CAL-A, CpLIP2 and its mutants were obtained from the
culture supernatant of transformed P. pastoris as described by Brunel
et al. [41] with the modifications described by Neang et al. [23]. The cul-
tivation, performed in 1 L or 3 L bioreactors, started by three successive
additions of glycerol (40 g·L−1) in batch mode and was followed by in-
duction by methanol feeding in fed-batch. The final extract was obtain-
ed after concentration and diafiltration of the supernatant by tangential
ultrafiltration (30 kDa) as described by Neang et al. [23].
Table 1
Production and characterization of recombinant CpLIP2, CAL-A and site directed mutants
of CpLIP2.
Lipid substrate
Enzyme
Ethyl oleate
Rapeseed oil
Detection of
recombinant
protein in culture
supernatantb
Specific
activityc
(U/mg)
% of the
activity
of CpLiP2
Activity on platea
2.8. Protein analysis
CpLIP2
CAL-A
S180A/C
D332A/N/S
H365A/N
D90N
C96A
N164K
Y179F
G181A
G181C
V213P
N215K
I231F
A333E
47.4
0.3
nd
nd
nd
10.1
0
25.2
18.0
91
1.5
26.8
2.6
24.8
13.0
0
100
1
+++
++
+
−
+
+
+
−
−
−
+
+
+
+
+
+
+
+
+
+
−
+
+
Concentrations of proteins in enzymatic extracts were determined
using the Bradford method [78] using pure, lyophilized CpLIP2 as
standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS/PAGE) was performed by the method of Laemmli [79], using
low-range protein markers (Bio-Rad SA, Ivry sur Seine, France), and
proteins were visualized by silver staining [80].
nd
nd
nd
21
0
53
38
193
9
57
5
52
27
0
+
+
++
++
+++
+
++
+
+++
++
−
2.9. Enzyme assays
Lipase activity detection was based on the rhodamine B plate assay de-
scribed in Section 2.6 and based on the method described by Kouker and
Jaeger [77] and modified by Brunel et al. [41]. Standard lipase activity was
determined by measuring the initial rate of ethyl oleate or ethyl laurate
hydrolysis at 30 °C, pH 6.5 as described by Neang et al. [23]. One enzyme
unit is the amount of enzyme that catalyzes the release of 1 μmol oleic
acid per min in these conditions. The study of the catalytic behavior
(alcoholysis and competitive hydrolysis) in the presence of ethyl oleate
with various methanol concentrations, and of methyl oleate with primary
alcohols (ethanol,1-propanol, 2-methyl-1-propanol and 1-butanol) and
C348A
P429F
I231F + P429F
6.2
5.8
13
12
+
+
a
Results of lipolytic activity assed on Rhodamine B plates (+++/++/+/−: big/
medium/small/no lipolysis halo surrounding the colony).
b
Results of protein visualization by SDS/PAGE of the final extracts obtained from the su-
pernatant of liquid cultures (+/−: visible/no band).
c
Specific activity is the initial specific hydrolysis rate of the corresponding lipid substrate
(nd: not detectable).

M. Subileau et al. / Biochimica et Biophysica Acta 1854 (2015) 1400–1411
1403
secondary alcohols (2-propanol, 2-butanol, and 3-pentanol) was per-
formed as previously described [23]. The transesterification ratio
corresponds to the relative initial rate of alcoholysis compared to the
total activity (alcoholysis and competitive hydrolysis). Specificity con-
stants 1/α were determined using an equimolar mixture of six saturated
ethyl esters as substrates (C8:0, C10:0, C12:0, C14:0, C16:0 and C18:0,
1.6 μmol each in 1 mL of reaction medium) as described by Neang et al.
[23]. All compounds were analyzed by GC-FID (flame ionization detector,
Hewlett-Packard 5890), after hexane extraction and silylation, as de-
scribed by Neang et al. [23] and the detection threshold corresponds to
0.1 μmol·mL−1 of product in the reaction medium. Only mutants that
showed a significant modification of catalytic behavior compared to
CpLIP2 are presented here.
sequence identities with CpLIP2 and low sequence similarity (b15%)
with other enzymes of known structure. An alignment of CpLIP2 and
CAL-A sequences is shown in Fig. 1. It is widely recognized that 3D struc-
tures are well conserved within protein families, even better than the
primary sequences are [81]. This assumption mainly relies on the hy-
pothesis of a common ancestor, which implies conservation of the
main structural characteristics, especially in the core of the protein. Se-
quence similarities between proteins thus strongly suggest structural
similarities. The CAL-A 3D structures (PDB ID: 2VEO and PDB ID:
3GUU), which were the only available templates, were thus used for
the construction of our model. Three 3D models of CpLIP2 were con-
structed, using the MUSTER [57], I-TASSER [57] and ROBETTA [59–61]
servers, and evaluated with Verify3D [62,63] and ERRAT [64] servers
(Supplementary data). MUSTER is a rather standard homology modeling
pipeline that generates models very close to the templates. I-TASSER is a
more elaborated process that e.g. includes ab initio loop reconstruction
and Monte-Carlo refinements. I-TASSER models may locally deviate
from the templates. ROBETTA is mainly dedicated to ab initio protein
modeling in the absence of close structural template. ROBETTA models
may significantly thus deviate from templates. It is worth noting that
3. Results & discussion
3.1. Modeling
Up to now, only the X-ray crystal structure of CAL-A has been solved
within the CAL-A lipase-like superfamily [54]. CAL-A exhibits 31% of
Fig. 1. Alignment of CpLIP2 and CAL-A sequences. Stars above the catalytic triad and the putative residues of the oxyanion hole are respectively highlighted in red and orange. Numberings
of the lid are highlighted in pink, and of main consensus sequences in purple.

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M. Subileau et al. / Biochimica et Biophysica Acta 1854 (2015) 1400–1411
the C-terminal part of the protein (from S284) exhibits a weak sequence
similarity with CAL-A and may not be correctly modeled.
embedded in the superfamily consensus sequence GYSGG and followed
by the YAPEL motif (Y196, A197, P198, E199, L200). Regarding more
precisely the residues supposed to play a key role in the catalytic activ-
ity, in the CAL-A structure, CAL-A_G185, located just next to the catalytic
serine, and CAL-A_D95 have been proposed as components of the
oxyanion hole [25,54]. In the CpLIP2 model, just after S180, residue
G181 could indeed be the first residue of the oxyanion hole (Fig. 2). Al-
though the acidic character of CAL-A_D95 makes it an unexpected can-
didate for the stabilization of the negative charge of the reaction
intermediate, this residue is highly conserved within the P. antarctica li-
pase A like superfamily (D90 in CpLIP2). More than stabilizing the tetra-
hedral intermediates, its acidic character has been hypothesized to
support a high pKa in this hydrophobic environment and therefore
favor the enzyme–substrate association [54]. In CpLIP2, the D90 residue,
situated less than 7 Å from S180 (Fig. 4), could also be an additional res-
idue of its oxyanion hole.
Looking at the global structure, the CpLIP2 3D model also suggests
some clusters of salt bridges located at the surface of the protein
(Fig. 2). These bridges occur between acidic (D or E) and basic (K, R or
H) residues. Two clusters are found in the N-terminal part of the en-
zyme: K6-D11-R32 for the first and K7-K93-D95 for the second. Other
bridges are formed opposite to the active site (K81-E173, D59-K139).
These salt bridges possibly participate in the stabilization of the protein
by increasing its rigidity. Other interesting features displayed by the
model are hydrophobic regions at the enzyme surface, as shown in
Fig. 3. The more hydrophobic parts (in red) are located around the ac-
tive site (Fig. 3A, C and D) and contain hydrophobic residues such as iso-
leucine, leucine, tyrosine, proline and phenylalanine. These regions
could be responsible for CpLIP2 high tendency to aggregate in solution,
because of hydrophobic interactions. The rest of the protein surface is
more hydrophilic (in blue, Fig. 3B) with numerous polar residues
found on the surface, and was more sensitive to proteases (see the lim-
ited proteolysis results in Section 3.2.1).
The CpLIP2 3D model that obtained the best structural evaluation
score was achieved with I-TASSER with an overall quality factor of
88.0 (ERRAT) and 0.72/0.06 max/min values (Verify 3D). For compari-
son, the crystal structure of CAL-A (PDB ID: 2VEOA) obtained 90.2 and
0.80/0.08 respectively. The I-TASSER 3D model of CpLIP2 is presented
in Figs. 2, 3 and 4. The CAL-A-based CpLIP2 model corresponds to a glob-
ular protein organized in two distinct domains (Fig. 2). The general
structure of CAL-A is conserved in CpLIP2 and corresponds to the de-
scription made by Ericsson et al. [54].
The main domain is a classical α/β hydrolase fold [82]. It is
complemented by a smaller and unusual well-defined lid made of 6 α
helices which is inserted between the β7 and β8 strands (Fig. 2). The
lid is connected to the rest of the protein by two hinges: on one side
the juxtaposed N215 and G310 loops that flank the lid sequence, and
on the other side a disulfide bond between the core and the lid (between
C96 and C269). Two other conserved cysteines (C348 and C393) are
linked by another disulfide bond in the α/β hydrolase domain.
The internal face of the lid, in contact with the active site, contains
hydrophobic residues while the outside part is of more hydrophilic
composition (Fig. 3). The active site has a cavity shape comprised be-
tween the β-sheet and the lid (Fig. 2). It contains the classical catalytic
triad (S180, D332, H365), the serine residue lying within the typical nu-
cleophilic turn located between the β6 strand and the α5 helix,
To go further, molecular docking of two substrates within the CpLIP2
model structure was performed (Fig. 4). The chosen substrates were
ethyl palmitoleate (C16:1EE), and ethyl palmitate (C16:0EE), for
which CpLIP2 displays different levels of activity [23]. For the calcula-
tions, an initial position of the substrate was assigned within a distance
of 25 Å from the catalytic S180. The resulting complexes (Fig. 4A) illus-
trate the substrate positioning in the enzyme. It appears that the only
docking position with a substrate orientation that allows interaction be-
tween the ester bond and the catalytic S is within the lid, where enough
space is found in the vicinity of the active site to welcome the acyl chain.
This position also favors the formation of a hydrogen bond between the
NH of G181 (putative first oxyanion hole residue) and the oxyanion in-
termediate. Moreover, the C16 carbon chains of the substrates fit well a
channel enclosed by hydrophobic residues in the lid (Fig. 4B2), and this
positioning is comparable to that of the polyethylene glycol molecule
that was trapped in the crystal structure of CAL-A (PDB ID: 2VEO). Inter-
estingly, the C16:0EE positioning appears less favorable than that of the
C16:1EE, with distances between the ester bond and the catalytic S180
of 3.90 and 3.06 Å, respectively. This is in good accordance with our pre-
vious experimental results, that showed that C16:0EE is hydrolyzed and
methanolyzed 25 times slower than C16:1EE by CpLIP2 [23]. Under the
lid, a hydrophobic valley (Fig. 3C) could welcome additional carbon
chains and appears well-adapted for the reception for example of acyl
chains from triacylglycerols explaining the broad spectrum of substrates
accepted by CpLIP2 [83].
Fig. 2. CpLIP2 3D model obtained by comparative modeling with the 3D structure of CAL-
A. The α/β hydrolase fold is organized around a β sheet (light blue) and α helix. The active
site contains the classical catalytic triad (yellow): S180, D332, H365. The lid (light pink) is
composed of 6 α-helix and is connected to the rest of the protein by two hinges: on one
side the juxtaposed N-terminal and C-terminal curls that flank the lid sequence, and on
the other side a disulfide bond between the core and the lid (between C96 and C269,
in cyan). The two other conserved cysteine residues build another disulfide bond in the
α/β hydrolase domain (between C348 and C396, in cyan). The putative first residue of
the oxyanion hole is in orange (G181). Salt bridges between acidic and basic residues
are located at the surface of the protein (dark green residues). Asparagine residues tested
for N-glycosylation are in aquamarine. Limited proteolysis cleavage sites (pink secondary
structures) are located at the periphery of the protein and in elbows between or at the ex-
tremity of secondary structures.
3.2. Experimental validation of the model
3.2.1. Limited proteolysis
Limited proteolysis on CpLIP2 was performed in order to validate
our 3D model. Structural domains of proteins are constituted of a spatial
assembly of secondary structures. In conditions of limited proteolysis,
the regions with higher accessibility (on the surface), lower stability

M. Subileau et al. / Biochimica et Biophysica Acta 1854 (2015) 1400–1411
1405
Fig. 3. Hydrophobicity at CpLIP2 surface. Hydrophilic amino-acids are in shades of blue, hydrophobic amino-acids are in shades of red. The surface of the residues submitted to site directed
mutagenesis is in orange. A: Hydrophobic side of the model, with access to the active site, as shown in Fig. 2. B: Hydrophilic site of the model, opposite to the active site. C: Surface of the
core protein, under the transparent lid, which outline is in green. The catalytic triad residues are written in blue. D: Inside surface of the lid in contact with the active site. The core of the
protein is transparent but outlined in green. The catalytic triad residues, located in the protein core, are in yellow sticks, before the lid.
Fig. 4. Docking of a molecule of ethyl palmitate (C16:0EE) or ethyl palmitoleate (C16:1EE) in CpLIP2. Residues of the catalytic triad are in yellow. CpLIP2 residues submitted to site directed
mutagenesis are in orange or magenta (I231 in the lid) and the substitution residues are in gray or purple for substitutions with CAL-A residues. Molecules of C16:0EE and C16:1EE are
respectively in dark green and light green. A: Docking of C16:0EE and C16:1EE in CpLIP2. Lid secondary structures are in light pink. The putative first oxyanion residue is in cyan. B1:
Docking of C16:1EE through CpLIP2 surfaces. Surface of the lid is in light pink and of the core protein is in tan. B2: Docking of C16:1EE viewed through CpLIP2 surfaces colored by hydro-
phobicity. Hydrophilic amino-acids are in shades of blue, hydrophobic amino-acids are in shades of red. Same view as in B1 but the surface is zoned around the C161EE, to 4 Å in the lid and
to 9 Å in the core protein.

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M. Subileau et al. / Biochimica et Biophysica Acta 1854 (2015) 1400–1411
A
B
100
90
80
70
60
50
40
30
20
10
0
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
1
2
3
4
0
1
2
3
4
Methanol concentration (M)
Methanol concentration (M)
Fig. 5. (A) Transesterification activity of CpLIP2, CAL-A and three mutants of CpLIP2 (Y179F, P429F and A333E). Values are expressed in percentage of total activity for each methanol con-
centration for CpLIP2 (◆), for CAL-A ( ), for Y179F ( ), for P429F (△) and for A333E (○). (B) Effect of methanol concentration on the total relative activity of the five enzymes with
increasing concentrations of methanol. Values for each enzyme are expressed relative to their specific activity in condition without alcohol. Reactions were performed during 15 min
at 30 °C, pH 6.5 in the presence of 10 mM of ethyl oleate.
and/or low interaction with the rest of the protein are the first to be hy-
drolyzed (turns and loops between secondary structures). Partial hy-
drolysis of the protein thus allows isolation and characterization of the
more stable secondary structures, particularly the β-sheets that are sta-
bilized by hydrogen bonds between the β-strands [84]. After optimiza-
tion of the conditions of proteolysis and Maldi-TOF analysis of the
obtained peptides, six main proteolysis sites were identified (K6/K7,
R32/N33, N164/D171, K202/N203, Y313/Q314 and K412/N413), giving
seven 25–47 kDa peptides. As shown in Fig. 2, many of the cleavages
are located near the protein termini, confirming that the N- and C-
terminal parts of CpLIP2 might be poorly structured. Three cleavage
sites (N164/D171, K202/N203, Y313/Q314) were located in more cen-
tral regions of the primary sequence, although in solvent-accessible
areas of the protein, in turns between or near ends of secondary struc-
tures. It is remarkable that most proteolysis sites were located on the
same side of the protein, in the most hydrophilic region, far away
from the active site (Figs. 2 and 3). The hydrophilic regions seem
more prone to proteolysis, probably because they are located on the
outer part of the CpLIP2 aggregates. On the contrary, the sequence be-
tween N203 and Y313, which includes the lid, is resistant to proteolysis.
No proteolysis has been observed inside predicted secondary structures
of the model, being only located in loops on the protein surface. Overall,
the results of the limited proteolysis of CpLIP2 are thus very well corre-
lated with the 3D model.
hydrolysis activity on ethyl oleate was observed (Table 1), but Y179F
was activated by the presence of methanol in the reaction medium,
resulting in a total activity similar to that of CpLIP2 in the presence of
3 M methanol (around 20 U·mg−1, Fig. 5). The change of polarity in-
duced by the Y179F mutation also resulted in an increase of the
transesterification ratio compared to the wild enzyme (Fig. 5). Indeed
in the presence of 0.3 M methanol, the specific transesterification ratios
displayed by Y179F and CpLIP2 were 87.5% and 74.1%, respectively
(Fig. 5). As shown in Fig. 4, Y179 is situated in a small cavity, just
under the lid. This cavity could be an access to the active site for the
acyl acceptor nucleophile, but also a trap in which the attacking nucleo-
phile would be more or less retained, by a mechanism of activation or
deactivation comparable to that described by Jiang et al. [85]. Yet, addi-
tional experiments with various primary and secondary alcohols as acyl
acceptor did not evidence any significant difference between CpLIP2
and the Y179F mutant (data not shown; see Fig. 7A for the CpLIP2 pro-
file). When the acyl donor substrate was ethyl laurate, this mutation
inducted a 40% increase of the specific activity compared to CpLIP2,
showing a difference in the selectivity between saturated and unsatu-
rated substrates. However, no change in the substrate specificity profile
on an equimolar mixture of C8 to C18 saturated ethyl esters was ob-
served between Y179F and the wild enzyme (data not shown; see
Fig. 6A for the CpLIP2 profile). This last result is in accordance with the
most probable position predicted by the docking calculation where
the Y179 does not appear in direct contact with the lipid substrate
(Figs. 3 and 4). The different selectivity observed in the previous exper-
iments also suggests that an alternative substrate docking location in
the vicinity of Y179 could exist where saturated and unsaturated sub-
strates would be discriminated.
The G181 residue, located at the entrance of a hydrophobic valley
(Fig. 3C), is supposed to play a central role in the oxyanion hole. This
possible first residue of the oxyanion hole should allow the stabilization
of the negatively charged intermediates by the formation of a hydrogen
bond with its main chain NH [54,85]. In our study, functional proteins
could be obtained with the G181A and G181C mutations. The G181A
mutation allowed a significant increase of specific activity while the
G181C mutation induced a strong decrease (Table 1). These results con-
firmed that the putative hydrogen bonding with the NH of the main
chain needed for the stabilization of the oxyanion is facilitated with a
small apolar glycine or alanine at this position, whereas the polar char-
acter of cysteine probably interferes with the stabilization. In addition
the need for a glycine or an alanine at this position may also be related
to the structural role of the narrow loop where the catalytic serine
stands (Fig. 2).
3.2.2. Site-directed mutagenesis
To further validate our model, variants of CpLIP2 were produced by
site directed mutagenesis and characterized (Table 1). The essential
functions of the catalytic triad (S180, D332, H365) and of the GXSXG
and YAPEL peptides were confirmed by their positioning within the
model, by their high conservation within the protein family and by
the result of their mutagenesis (deleterious mutations: S180A/C,
D332A/N/S, H365A/N, see Table 1 and Supplementary materials). Addi-
tional substitutions of residues located in the vicinity of the modeled ac-
tive site were performed. Besides the catalytic triad, three residues of
CpLIP2 were modified because they appeared important for the catalyt-
ic activity.
The importance of Y179 and G181, included in the highly conserved
GYSGG motif of this enzyme superfamily was investigated, as well as
that of the D90. Neither Y179A nor Y179C mutations allowed the signif-
icant production of a functional protein, but the mutant Y179F, very ac-
tive, could be produced and characterized (Table 1). These first results
tend to show that an aromatic residue seems to be needed at this posi-
tion. With the mutant Y179F, compared to CpLIP2, a loss of 63% of the

M. Subileau et al. / Biochimica et Biophysica Acta 1854 (2015) 1400–1411
1407
A
B
C
D
E
F
Fig. 6. Specificity constants (1/α) for hydrolysis of ethyl esters of saturated FA C8–C18 catalyzed by CpLIP2 (A), CAL-A (B), and the mutants of CpLIP2: V213P (C), I231F (D), P429F (E) and
I231F/P429F (F). Reactions were performed during 15 min at 30 °C, pH 6.5 in the presence of 10 mM of ethyl esters mixture (1.6 mM of each ester).
Next, the role of an acidic residue in the putative oxyanion hole was
investigated with the mutation D90N. In CAL-A, saturation mutagenesis
of the corresponding CAL-A_D95 did not allow the production of any
functional protein and confirmed the crucial role of this residue for the
catalytic activity of CAL-A [86]. In CpLIP2 the D90N mutation allowed
the production of a functional protein, although not highly active on
ethyl oleate (loss of 78% of the hydrolysis activity, Table 1). Yet, com-
pared to CpLIP2, an increase of 25% of the activity on ethyl laurate
could be observed with the D90N mutation, but no modification of the
substrate specificity profile or reaction specificity (transesterification ra-
tios) could be observed compared to CpLIP2. Thus, the role of the D90 in
the oxyanion hole of CpLIP2 remains unclear. In our model, D90 is not di-
rectly oriented toward the catalytic serine contrary to its homologous
CAL-A_D95. It is more buried in the core protein and may not play the
same role as CAL-A_D95. Further mutations of other residues could
thus be conducted for the elucidation of the CpLIP2 oxyanion hole
structure.
This disulfide bridge, acting as a hinge for the lid, is thus very impor-
tant for the structure of the active enzyme. The second disulfide bridge,
between C348 and C393 is even more essential. Indeed, the C348A mu-
tation did not allow the production of the corresponding protein
(Table 1). This confirms that this second disulfide bridge, located oppo-
site to the active site between a loop and a helix, also plays a key role in
the protein structure (Fig. 2).
Among the two potential N-glycosylation sites identified using the
Prosite database [87] (N164 and N215, Fig. 2), only the N164K substitu-
tion provoked a mass loss, resulting in a protein with the molecular
weight expected from the protein sequence. This mutation, located on
the surface of the modeled protein, induced an average 50% decrease
of the specific activity, but no other modification. The N215K substitu-
tion resulted in no mass loss but induced an important loss of activity,
probably due to the position of this residue in a critical place at the N-
terminal loop of the lid hinge.
It can be concluded that the effect of the mutations described here is
well correlated with their situation on the 3D model of CpLIP2.
3.2.2.1. Key residues for protein structure. On the model, C96 and C269
build a disulfide bridge between the protein and the lid (Fig. 2). The
C96A mutation allowed the detection of a minor activity on plates and
a small production of the recombinant protein (Table 1). Nevertheless,
no activity could be quantified with this recombinant protein.
3.2.2.2. Variations between CpLIP2 and CAL-A in the vicinity of the active
site. To go further into the elucidation of CpLIP2 specificities, after a closer
look at the residues differing between CpLIP2 and CAL-A in the vicinity of
the active site, four additional mutation sites were chosen: A333, V213,

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M. Subileau et al. / Biochimica et Biophysica Acta 1854 (2015) 1400–1411
I231, and P429. Indeed, even if CpLIP2 and CAL-A belong to the same su-
perfamily and have significant sequence and structure homologies, only
CpLIP2 exhibits an exceptional alcoholysis activity in biphasic aqueous
media [23]. With the aim to better understand this acyltransferase reac-
tion specificity, we chose to substitute the selected residues of CpLIP2 for
the corresponding amino-acids of the CAL-A structure. Five mutants
were thus built: A333E, V213P, I231F, P429F and a double mutant
I231F/P429F.
the specific hydrolysis activity on ethyl oleate and an increase of 43%
of the specific hydrolysis activity on ethyl laurate, compared to CpLIP2.
With the methanol concentrations tested, no significant change in the
transesterification ratios was observed. Yet, additional experiments
with various primary and secondary alcohols have shown that the
I231F mutation in CpLIP2 provoked a significant change in the acyl ac-
ceptor specificity (Fig. 7). Indeed, while the transesterification ratios ob-
tained with CpLIP2 and the other mutants were comparable for primary
and secondary alcohols for a same relative thermodynamic activity
(aalcohol / [aalcohol + aw], see [23]), the I231F mutation induced a strong
decrease of the transesterification ratios obtained with the secondary
and branched alcohols (isobutanol, propan-2-ol, butan-2-ol, pentan-3-
ol; Fig. 7). This mutation thus induces selectivity toward the acyl accep-
tor, with a final profile similar to that of the AflaL0 lipase described in a
previous work [23]. Besides, the specificity profile on saturated ethyl es-
ters was also modified by the I231F mutation in favor of shorter acyl
chains (Fig. 6). Indeed, the highest specificity constant was still obtained
with C12 ethyl ester, but C8 and C10 were also preferential substrates
(1/α higher than 0.8) (Fig. 6). The replacement of an isoleucine with a
phenylalanine residue at this position does not change the global hydro-
phobicity, but the lower flexibility of phenylalanine finally appears
(i) unfavorable to bulky alcohols as acyl acceptor and (ii) advantageous
for short chain esters as acyl donors. On the CpLIP2 3D model, the posi-
tion of I231 appears indeed critical for the access of the substrates to the
active site (Fig. 4B1). In Fig. 4A, a clash between the phenylalanine (mu-
tation I231F) and the C16 lipid substrate docked in the CpLIP2 3D model
is even visible, but this mutation probably induces structural rearrange-
ments which are not observable on this 3D model.
CAL-A_F431 has been described as a crucial component for the activ-
ity of CAL-A, playing the role of a hydrophobic bottleneck at the entrance
of the substrate tunnel that could limit the range size of the substrates
[86,88]. In CpLIP2, the P429F mutation allows the production of a func-
tional enzyme that exhibited a loss of specific hydrolysis activity of 87%
on ethyl oleate and of 25% on ethyl laurate. The specific activities of the
double mutant enzyme I231F/P429F were in the same range as that of
the single P429F mutant (Table 1). In the presence of methanol, the
transesterification activity of the P429F mutant was inferior to that of
the wild type CpLIP2, with respectively transesterification ratios of 58%
and 74% in the presence of 0.3 M methanol (Fig. 5). The I231F/P429 dou-
ble mutant has an intermediary behavior with a transesterification ratio
of 66% in the presence of 0.3 M methanol (Fig. 5). With other primary
and secondary alcohols, the acyltransfer activity of P429F was compara-
ble to that of CpLIP2 but the double mutant I231F/P429F exhibited the
same catalytic specificity toward linear acyl acceptors as I231F. The
P429F mutation induced an inversion of preference for C10 instead of
C12 ethyl ester in competitive conditions, and a slight increase of the
specificity constant for C8 (Fig. 6). The double mutant enzyme exhibited
a specificity profile comparable to that obtained with the I231F mutation
(Fig. 6). In P429F, the replacement of a small proline into a large hydro-
phobic phenylalanine was expected to have a more important effect on
substrate specificity. It appears that the main consequence was in fact
an important decrease of the specific activity, maybe because, like in
CAL-A, the phenylalanine residue creates a bottleneck that limits the ac-
cessibility of the substrate to the active site. In the CpLIP2 3D model, P429
does not appear to be in direct contact with the lipid substrate (Fig. 4)
but it is located at a possible entry of the active site, in contact with the
lid (Fig. 3A and D). Its location on the lid-like C-terminal flap could also
play a role in the interfacial activation of the enzyme, and again on the
access of the substrate to the active site.
The A333E mutation, located just after the catalytic acid, appeared
quite challenging, with the change of a small hydrophobic residue into
a large acidic one. However, this mutation allowed the production of
an active protein which, compared to CpLIP2, exhibited losses of 73%
and 49% of specific hydrolysis activity on ethyl oleate and ethyl laurate,
respectively. No modification of the specificity profile on saturated sub-
strates could be observed, but the A333E mutation induced a decrease
of the transesterification ratio (Fig. 5). Indeed, in the presence of 0.3 M
methanol, alcoholysis catalyzed by the A333E mutant represented only
45% of the total activity against 74% with CpLIP2. The A333 is situated
just at the entrance of the active site, and is in contact with the solvent
(Fig. 3A). The presence of an alanine residue at this position is thus favor-
able to the reaction with acyl acceptors less polar than water. It is possi-
ble that, in CAL-A, the acidic character of this residue favors the
positioning of water molecules in the vicinity of the active site by activa-
tion of the attacking water molecule as suggested by Jiang et al. [85].
Looking at the other lipases and lipases/acyltransferases of this family
characterized in the same conditions [22,23], CpLIP2, which exhibits
the best acyltransferase property, is the only one that possesses an ala-
nine at this position. AflaL0, which catalyzes low acyltransfer reaction,
possesses a glutamic acid like CAL-A [23]. Interestingly, the two other
acyltransferases CtroL0 and CaLIP4 have a glutamine at this position, a
polar but not charged residue, less favorable to water than the acid but
still more polar than alanine. However, this position is not the only one
involved in the acyltransferase character of CpLIP2 as CtroL0, CaLIP4
and the A333E mutant all display a superior transesterification ratio
than CAL-A, as shown in Fig. 5A.
The V213P mutation, at a distance of 5 Å to the catalytic S180, is in
contact with the lid (Fig. 3C) and the lipid substrate (Fig. 4). The
corresponding CAL-A_P215 is supposed to play a role in the positioning
of the acyl moiety of the substrate [88]. If this mutation in CpLIP2
did not induce a significant change in the reaction specificity (the
transesterification ratios are comparable to that of CpLIP2, data not
shown), the loss of specific hydrolysis activity on ethyl oleate was only
of 43%. More surprisingly, the V213P mutation allowed an 84% increase
of the specific hydrolysis activity on ethyl laurate. In addition the speci-
ficity profile of the V213P mutant on saturated ethyl esters also differed
from that of CpLIP2 (Fig. 6A and C). The specificity constants show that
the V213P mutant has a preference for shorter acyl chains than CpLIP2,
with a specificity toward C10 (preferred substrate) and C8 ethyl esters
(1/α of 0.78 compared to 0.40 obtained with CpLIP2). Thus, the substitu-
tion of the valine of CpLIP2 into the proline of CAL-A induced the produc-
tion of a mutant enzyme with unique specificity profile compared to
CpLIP2 or CAL-A (Fig. 6). This confirms that this area plays a role in the
substrate specificity of the enzyme. On the CpLIP2 3D model, V213 ap-
pears at the entry of a hydrophobic valley (Fig. 3C) that could indeed
welcome fatty acid chains in a more open conformation of the enzyme.
It is also the only mutated residue that can be seen from the “hydrophilic
side” of the enzyme (Fig. 3B).
Finally, the two additional mutations I231F and P429F were chosen
because these residues are positioned at a possible entry of the active
site, in the lid for I231 (Figs. 3 and 4). Moreover, the corresponding res-
idues in CAL-A (CAL-A_F233 and CAL-A_P431, respectively) have al-
ready been described as playing a role in the substrate specificity of
CAL-A [86,88,89]. CAL-A_F233 has been described as a hot spot in CAL-
A, especially for enhancement of the substrate cavity space and of
the enantioselectivity [86,88,89]. The CpLIP2 mutation I231F allowed
the production of a functional enzyme that exhibited a loss of 46% of
Finally, apart from the A333E mutation that induced an important
loss of the transesterification activity compared to CpLIP2, the substitu-
tions of CpLIP2 residues for those of CAL-A did not convert the specific-
ities of CpLIP2 into those of CAL-A. They induced some changes in
substrate specificity, particularly with the V213P and I213F mutations
that are the closest to the docked substrates (Fig. 4), but the profiles ob-
tained were different from those of CpLIP2 or CAL-A. Logically, the

M. Subileau et al. / Biochimica et Biophysica Acta 1854 (2015) 1400–1411
1409
Fig. 7. Relationships between the relative thermodynamic activity of alcohols (aalcohol / (aalcohol + aw)) and the transesterification activity at two alcohol concentrations (0.03 and 0.3 M) for
CpLIP2 (A) and its mutant I231F (B). Full symbols correspond to primary alcohols and empty ones to secondary alcohols. Reactions were performed at 30 °C, pH 6.5 in the presence of
10 mM C18:1 ME and 0.03 M or 0.3 M of alcohol. Thermodynamic activities were estimated using UNIFAC-LLE taking into account the presence of alcohol, water and ester in a biphasic
system at 30 °C. With the mutant I231F no activity was detected with 0.3 M of isobutanol.
substitution of short hydrophobic residues by phenylalanines in the vi-
cinity of the active site (I231F and P429F) tends to be unfavorable to
long chain fatty esters. In CAL-A, the presence of these phenylalanines
(CAL-A_F233 and CAL-A_F431) could be somewhat responsible for its
low specific activity, particularly on unsaturated substrates compared
to CpLIP2 as previously described [23]. Nevertheless, other residues
must be involved in the substrate accessibility to active site because
(i) despite the presence of phenylalanines, CAL-A exhibits a preference
for the medium-length C14 ethyl ester (Fig. 6) and (ii) CtroL0, that pos-
sesses isoleucine and proline in the same position as CpLIP2, has a
marked preference for the longer C16 ethyl esters [23].
proteolysis. Limited proteolysis study resulted in hydrolysis sites found in
regions of low structure at the periphery of the protein 3D model. Exper-
imental data obtained after site-directed mutagenesis were also consis-
tent with features of the model. The substitution of G181, the putative
first residue of the oxyanion hole, by alanine resulted in a fully active en-
zyme but its replacement by cysteine provoked an important loss of activ-
ity. Besides, the proposed role of D90 in the stabilization of the tetrahedral
oxyanion intermediate remains unconfirmed in the case of CpLIP2. The
Y179F substitution, embedded into the highly conserved GYSGG motif,
allowed the production of a functional mutant enzyme with better
transesterification activity than CpLIP2. The comparison with the CAL-A
3D structure was then helpful for the rational choice of mutagenesis
sites. The effects of these mutations on CpLIP2 were well correlated
with the location of the corresponding residues on the 3D model. The
A333E and P429F mutations, in contact with the solvent (Fig. 3A), induce
a change in the reaction specificity of the enzyme with a decrease of the
transesterification activity, which brings these mutants closer to CAL-A.
The V213P, I231F and P429F mutations induce a change in substrate spec-
ificity by favoring the shorter chain saturated ethyl esters compared to
CpLIP2 but also to CAL-A. Besides, with the I231F mutation, a significant
decrease of the transesterification ratios was observed only with the
branched alcohols, suggesting a crucial role of this position for the entry
or positing of the acyl acceptor.
Despite their similarity of structure, CpLIP2, CAL-A and the CpLIP2
mutants thus exhibit differences in their catalytic properties. The sub-
strate and reaction specificities of CpLIP2 and CAL-A probably rely on a
combination of several structural elements and rearrangements that
punctual mutations cannot fully elucidate. Yet, the three-dimensional
model of CpLIP2, in comparison with CAL-A 3D structure, is now useful
to identify structural determinants for the peculiar features of CpLIP2
and of this original family of enzymes. The model is currently used for
targeted modifications of the enzyme to change its substrate specificity
and improve its thermostability.
3.3. Lid(s) and interfacial positioning
Further away from the active site, some differences between CpLIP2
and CAL-A also appear in the internal part of the lid, in contact with
the active site. The characteristics of the lid, hydrophobic inside and
more polar outside, strongly suggest a possible role in the interfacial po-
sitioning of CpLIP2 (Fig. 3). Both hinges and additional contact zones
could limit the mobility of the lid. Therefore, the interfacial positioning
may not induce a total swing of the lid, but rather a relaxation of some
of the α helices that could allow the penetration of the substrate into
the active site like in Thermomyces lanuginosa lipase [90]. In addition to
residues such as P429, the global polarity of the rest of the protein prob-
ably governs the orientation of the enzyme at the lipid/water interface. In
the CAL-A structure, the lid and the C-terminal peptide have been de-
scribed as the most original but also unconserved regions of CAL-A struc-
ture, compared to its superfamily [25]. The hydrophobic patches in these
regions include aromatic residues that could reorient to meet the lipid
interface, such as in C. rugosa lipase [56] which binding site is similar to
that of CAL-A [25]. The C-terminal peptide could also act as an active-
site flap and play a role in the solvent accessibility to the active site. How-
ever, since these regions could have been inaccurately modeled in
CpLIP2 because of the lack sequence similarity with CAL-A, further pro-
tein engineering strategies should be conducted to better characterize
and understand their functional role. Indeed, low evaluation scores
with both ERRAT and Verify 3D were observed for these segments
(Supplementary data). Moreover, as they are less structured and there-
fore less stable, it would be interesting to know if they are also more
vulnerable to other aggressions such as changes of temperature or pH.
Author contributions
The manuscript was written through contributions of all authors.
Funding sources
A. H. Jan was supported by BASF (n°22008910) within the Metaglyc
2 project.
4. Conclusion
H. Nozac'h was supported by a grant from the French Ministry of Re-
search, Education and Technology.
The homology between CpLIP2 and CAL-A allowed the computation of
a 3D model based on crystal structures of CAL-A. The conservation of the
active site region between the two enzymes supports the hypothesis of a
classical serine hydrolase catalytic mechanism for CpLIP2 based on the
catalytic triad S180, D332 and H365. The CpLIP2 model was confronted
to experimental data obtained after site-directed mutagenesis and limited
Abbreviations
CpLIP2 lipase/acyltransferase from Candida parapsilosis
CAL-A
lipase A from Pseudozyma (Candida) antarctica

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Acknowledgment
Modeling was realized with the help of Dr Gilles Labesse, A.B.C.I.S.
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Appendix A. Supplementary data
(A) Evaluation scores with ERRAT and Verify 3D of different CpLIP2
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(B) Additional production and characterization data on CpLIP2 mutants.
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