Structural insights into the catalytic mechanism of lovastatin hydrolase

Article abstract of DOI:10.1016/S0021-9258(17)49914-5

The lovastatin hydrolase PcEST from the fungus Penicillium chrysogenum exhibits enormous potential for industrial-scale applications in single-step production of monacolin J, the key precursor for synthesis of the cholesterol-lowering drug simvastatin. This enzyme specifically and efficiently catalyzes the conversion of lovastatin to monacolin J but cannot hydrolyze simvastatin. Understanding the catalytic mechanism and the structure-function relationship of PcEST is therefore important for further lovastatin hydrolase screening, engineering, and commercial applications. Here, we solved four X-ray crystal structures, including apo PcEST (2.3 ?), PcEST in complex with monacolin J (2.48 ?), PcEST complexed with the substrate analog simvastatin (2.4 ?), and an inactivated PcEST variant (S57A) with the lovastatin substrate (2.3 ?). Structure-based biochemical analyses and mutagenesis assays revealed that the Ser57 (nucleophile)-Tyr170 (general base)-Lys60 (general acid) catalytic triad, the hydrogen-bond network (Trp344 and Tyr127) around the active site, and the specific substrate-binding tunnel together determine efficient and specific lovastatin hydrolysis by PcEST. Moreover, steric effects on nucleophilic attack caused by the 2',2-dimethybutyryl group of simvastatin resulted in no activity of PcEST on simvastatin.Onthe basis of structural comparisons, we propose several indicators to define lovastatin esterases. Furthermore, using structure-guided enzyme engineering, we developed a PcEST variant, D106A, having improved solubility and thermostability, suggesting a promising application of this variant in industrial processes. To our knowledge, this is the first report describing the mechanism and structure-function relationship of lovastatin hydrolase and providing insights that may guide rapid screening and engineering of additional lovastatin esterase variants.

Full text of DOI:10.1016/S0021-9258(17)49914-5

cro
ARTICLE
Structural insights into the catalytic mechanism of lovastatin
hydrolase
Received for publication, November 15, 2019, and in revised form, December 7, 2019 Published, Papers in Press, December 15, 2019, DOI 10.1074/jbc.RA119.011936
X Yajing Liangठand X Xuefeng Luद1
From the ‡Key Laboratory of Biofuels and §Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of
Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China and the ¶Laboratory for Marine
Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266003, China
Edited by Joseph M. Jez
The lovastatin hydrolase PcEST from the fungus Penicillium
chrysogenum exhibits enormous potential for industrial-scale
applications in single-step production of monacolin J, the key
precursor for synthesis of the cholesterol-lowering drug simvas-
tatin. This enzyme specifically and efficiently catalyzes the con-
version of lovastatin to monacolin J but cannot hydrolyze
simvastatin. Understanding the catalytic mechanism and the
structure–function relationship of PcEST is therefore impor-
tant for further lovastatin hydrolase screening, engineering, and
commercial applications. Here, we solved four X-ray crystal
structures, including apo PcEST (2.3 Å), PcEST in complex with
monacolin J (2.48 Å), PcEST complexed with the substrate ana-
log simvastatin (2.4 Å), and an inactivated PcEST variant (S57A)
Simvastatin, a derivative of the fungal polyketide lovastatin,
is a major drug used to treat hypercholesterolemia. It is indus-
trially produced through three steps: 1) Aspergillus terreus fer-
mentation to produce lovastatin; 2) lovastatin alkaline hydroly-
sis to monacolin J; and 3) chemical or biological transformation
of monacolin J to produce simvastatin (Fig. 1) (1–5). After the
above steps, the 2-methylbutyryl side chain of lovastatin will be
replaced by the 2,2-dimethylbutyryl side chain, thereby pro-
ducing simvastatin. However, the multistep alkaline hydrolysis
process to produce monacolin J is complicated, expensive, and
environmentally unfriendly. Enzymatic synthesis using a spe-
cific lovastatin hydrolase is one of the alternative methods for
the green production of monacolin J. In addition to their impor-
with the lovastatin substrate (2.3 Å). Structure-based biochem- tant role in green processes, specific lovastatin hydrolases with-
ical analyses and mutagenesis assays revealed that the Ser57 out simvastatin hydrolysis activity are also industrially impor-
(nucleophile)–Tyr170 (general base)–Lys60 (general acid) cata- tant because of their use in removing the residual lovastatin in
lytic triad, the hydrogen-bond network (Trp344 and Tyr127
)
simvastatin samples.
around the active site, and the specific substrate-binding tunnel
together determine efficient and specific lovastatin hydrolysis
by PcEST. Moreover, steric effects on nucleophilic attack caused
by the 2؅,2-dimethybutyryl group of simvastatin resulted in no
activity of PcEST on simvastatin. On the basis of structural com-
parisons, we propose several indicators to define lovastatin
esterases. Furthermore, using structure-guided enzyme engi-
neering, we developed a PcEST variant, D106A, having im-
proved solubility and thermostability, suggesting a promising
application of this variant in industrial processes. To our knowl-
edge, this is the first report describing the mechanism and
structure–function relationship of lovastatin hydrolase and
providing insights that may guide rapid screening and engineer-
ing of additional lovastatin esterase variants.
The discovery of lovastatin hydrolases with industrial appli-
cation potential has been very slow. As early as 1997, a specific
lovastatin esterase (unpublished protein sequence) was isolated
and purified by Merck (6). Subsequently, this strain was used to
establish a fermentative conversion system for monacolin J pro-
duction (7). In 2004, Morgan reported another specific lovasta-
tin esterase, EcBla4 (8). However, Km values of up to 200–300
␮M lovastatin restrict the industrial use of these lovastatin
esterases to produce monacolin J. In 2017, we reported that a
specific lovastatin esterase from Penicillium chrysogenum
(called PcEST) catalyzed the single-step conversion of lovasta-
tin to monacolin J with a 232-fold higher catalytic efficiency
than EcBla4 (Table 1) and simultaneously displayed no activity
against simvastatin. Further, we achieved ϳ95% conversion by
heterologously expressing PcEST in an industrial A. terreus
strain (2). Recently, we further reduced the lovastatin residual
using a directed evolutionary PcEST mutant, Q140L, which
This work was supported by National Natural Science Foundation of China presents 2.2-fold improved solubility, 1-fold enhanced catalytic
10
Grant 31700051 (to Y. L.), Natural Science Foundation of Shandong Prov-
ince Grant ZR2017BC065 (to Y. L.), Science and Technology Service Net-
work Initiative of the Chinese Academy of Sciences Grant KFJ-STS-ZDTP-
efficiency, and 3 °C increased T50 over the purified WT
PcEST (9). All of these results clearly suggest the huge industrial
031, and a Shandong Taishan Scholarship (to X. L.). The authors declare application potential of PcEST.
that they have no conflicts of interest with the contents of this article.
This article contains Tables S1 and S2 and Figs. S1–S3.
Bioinformatics analyses showed that the function-identified
proteins with the top three sequence identities (ϳ36–40%)
with PcEST are LovD from A. terreus, mokF from Monascus
pilosus, and mlcH from Penicillium citrinum (Fig. S1). Interest-
ingly, all of them are acyltransferases, which are mainly respon-
sible for the transacylation reaction in different statin biosyn-
The atomic coordinates and structure factors (codes 6KJC, 6KJE, 6KJF, and 6KJD)
have been deposited in the Protein Data Bank (http://wwpdb.org/).
1 To whom correspondence should be addressed: Qingdao Institute of Bio-
energy and Bioprocess Technology, Chinese Academy of Sciences, 189
Songling Rd., Qingdao 266101, China. Tel.: 86-532-80662712; E-mail: lvxf@
qibebt.ac.cn.
This is an Open Access article under the CC BY license.
J. Biol. Chem. (2020) 295(4) 1047–1055 1047
© 2020 Liang and Lu. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanism and engineering of lovastatin esterase PcEST
Figure 1. The industrial process for simvastatin production (2). Simvastatin only has one more methyl group than lovastatin. The 2-methylbutyryl side
chain of lovastatin and 2Ј2-dimethybutyryloxy side chain of simvastatin are colored red and blue, respectively. The biological transformation route of simvas-
tatin is labeled by a green arrow.
Table 1
Kinetic analysis with lovastatin of PcEST and its mutants
The values shown represent the averages of at least two repetitive measurements plus standard deviation.
Enzyme
Km
␮M
kcat
min
k
cat/Km
Reference
Ϫ1
Ϫ1
Ϫ1
min ␮M
PcEST
EcBla4
LovD
39.4 Ϯ 4.3
237.5 Ϯ 15.5
560 Ϯ 50
194.6 Ϯ 5.5
5.0 Ϯ 0.1
4.9 Ϯ 0.4
Huang et al. (2) (37°)
0.0211 Ϯ 0.0009
0.0004 Ϯ 0.000016
37.38 Ϯ 3.50
0.21 Ϯ 0.01
204.1 Ϯ 5.93
8.93 Ϯ 0.35
1.26 Ϯ 0.08
175.8 Ϯ 8.99
158.7 Ϯ 9.0
Xie et al. (10) (25°)
PcEST
Y127F
W344F
D106A
D131A
5.46 Ϯ 0.67
218 Ϯ 23.15
151.4 Ϯ 28.69
8.67 Ϯ 2.06
9.11 Ϯ 2.09
This work (30°)
0.04 Ϯ 0.003
0.008 Ϯ 0.0010
20.28 Ϯ 3.78
17.42 Ϯ 3.01
thetic pathways. LovD is a well-studied enzyme with the highest and the S57A inactivation mutant with the substrate lovastatin.
sequence identity (up to 40%) with PcEST. This enzyme mainly
catalyzes the transfer of the 2-methylbutyl side chain to mona-
colin J for the synthesis of lovastatin. In addition to acyltrans-
ferase activity, LovD also reversely catalyzes the lovastatin hy-
Structure-based biochemical analyses and mutagenesis assays
revealed the efficient and specific lovastatin hydrolysis mecha-
nism and identified why PcEST has no activity toward simvas-
tatin. In addition, based on structural comparisons, several
indicators were proposed for the screening of subsequent lov-
astatin esterases. Furthermore, we successfully developed a sol-
ubility and thermostability improved D106A mutant through
structure-guided enzyme engineering, suggesting a promising
application of this variant in industrial processes.
Ϫ1
drolysis reaction with kcat and Km values of 0.21 Ϯ 0.01 min
and 0.56 Ϯ 0.05 mM, respectively (10), which represents an
ϳ10Ϫ5-fold catalytic efficiency of PcEST (Table 1). However,
the specific lovastatin esterase EcBla4 shares only 16.8%
sequence identity with PcEST. Moreover, all of these enzymes
contain the conserved SXXK motif (where X represents any
residue), which is important for catalysis in many serine-based
enzymes (11). These results mean that screening for new and
efficient lovastatin hydrolases through homologous sequence
alignment methods is relatively inefficient and uneconomical.
However, to date, the specific catalytic mechanism and
structure–function relationships of lovastatin hydrolase have
not been reported. Thus, it is important to clarify the specific
and efficient lovastatin hydrolysis mechanism of PcEST to
enable further rapid screening, protein engineering, and com-
mercial application of lovastatin esterases.
Results and discussion
Sequence analysis of PcEST
First, the sequence alignment of 15 enzymes including
PcEST, EcBla4, and members of the carboxylesterases family,
acyltransferase family, and ␤-lactamase family was performed
(Fig. S1). Although PcEST shares only 16.8% sequence identity
with EcBla4, both enzymes contain the conserved SXXK motif
of ␤-lactamases or family VIII carboxylesterases. In this motif,
the serine acts as a nucleophile and initiates hydrolysis (12). In
addition to the SXXK motif, PcEST has two motifs characteris-
tic of family VIII carboxylesterases, YXX and WGG (absent in
In the present study, we solved four crystal structures of the
lovastatin hydrolase PcEST, including apo PcEST, PcEST in
complex with monacolin J, PcEST in complex with simvastatin, EcBla4) (11). However, the highly conserved YXN and K(T/S)G
1048 J. Biol. Chem. (2020) 295(4) 1047–1055

Mechanism and engineering of lovastatin esterase PcEST
Figure 2. Mutagenesis and structural analyses of PcEST. A, lovastatin hydrolysis activity of PcEST mutants in vitro. The final concentration of all mutants is
100-fold greater than the WT PcEST control. The substrate lovastatin acid is abbreviated as LVA, and the product monacolin J acid is abbreviated as MJA.
Moreover, themonacolinJacidpeaksinmutantsK60SandK60AHPLCcurvesaremarkedbyasterisks. B, cartoonrepresentationoftheapoPcESTstructure. Two
domains are colored separately in lime green and light pink. The possible Ser57–Lys60–Tyr170 catalytic triad is shown as sticks with red. C, superimposition of the
apo PcEST structure and S57A–lovastatin complex. S57A is presented with blue, and lovastatin is shown with yellow sticks. The conformational change in PcEST
loop 238–245 after statin binding is marked with a red arrow. D–G, zoomed view of the apo PcEST and three PcEST–ligand complex catalytic centers. The
2Fo Ϫ Fc densities for the active site are contoured in blue at 1.5 ␴. Hydrogen bonds are indicated by black dashed lines, and H2O is shown as a red ball. The
distances between key atoms that do not form hydrogen bonds are labeled in red.
motifs found in ␤-lactamases are not present in PcEST (13). complex, were solved. The structure resolutions of apo PcEST,
These sequence analyses imply that PcEST is a family VIII car-
boxylesterase, with Ser57 as a putative nucleophile. Elimination
of Ser57 nucleophilic activity in the S57A mutant completely
abolished lovastatin hydrolysis, supporting the key role of Ser57
in PcEST-mediated lovastatin hydrolysis (Fig. 2A).
S57A–lovastatin complex, PcEST–monacolin J complex, and
PcEST–simvastatin complex are 2.3, 2.3, 2.48, and 2.4 Å,
respectively. The data collection and refinement statistics are
provided in Table S2.
Like other reported family VIII esterases (11, 14, 15), the
PcEST proteins in all four structures adopt a two-domain mod-
ular structure containing a typical ␣/␤ hydrolase domain (res-
idues 1–75 and 189–399) and a small ␣-helical like domain
(residues 76–188). A highly hydrophobic substrate-binding
Overall structure of PcEST
To gain deeper insights into the structure–function relation-
ships of PcEST, four crystal structures, including apo PcEST,
PcEST–monacolin J complex, PcEST-simvastatin complex,
and the S57A inactivation mutant with the substrate lovastatin pocket is formed at the two-domain interface with the con-
J. Biol. Chem. (2020) 295(4) 1047–1055 1049

Mechanism and engineering of lovastatin esterase PcEST
Figure 3. Verification of two nucleophiles in PcEST-mediated lovastatin hydrolysis. A, active site superimposition of the apo PcEST (left panel) or S57A–
LVA complex (right panel) and the DFP-bound esterase EstB. PcEST or S57A and EstB are separately colored white and blue. The catalytic triads are shown as
sticks, andH2OinapoPcESTisshownasaredball. DFP(orange)overlapswiththe2-methylbutyrylsidechainoflovastatin(yellow)andthepossiblecatalyticH2O
in PcEST. B, H218O isotope labeling assay using GC-MS detection.
served 57SATK60 motif in the floor of the interface depression ther, Ser57 forms hydrogen bonds with Lys60 and Tyr170
,
(Fig. 2, B and C).
completing the PcEST catalytic triad (Fig. 2, D and E). More-
over, this putative Ser57–Lys60–Tyr170 catalytic triad com-
pletely overlaps the Ser75–Lys78–Tyr181 catalytic triad of ester-
ase EstB (PDB2 code 1CI9) (11), suggesting these residues
indeed comprise the catalytic site (Fig. 3A). Furthermore, the
phosphoryl group of the diisopropyl-fluorophosphate (DFP, an
inhibitor of serine hydrolases using its phosphoryl group cova-
lent attachment to catalytic serine) in the EstB structure
matches well with the carbonyl group of lovastatin (Fig. 3A).
These structural analyses, combined with abolished S57A
activity, demonstrate the nucleophilic function of Ser57.
Possible catalytic mechanism in PcEST-mediated lovastatin
hydrolysis
We then examined the PcEST-mediated lovastatin hydroly-
sis mechanism. The general mechanism of family VIII esterases
is thought to occur via two successive nucleophilic attacks by
the serine in the Ser–Lys–Tyr catalytic triad and a water mole-
cule (11). In fact, this mechanism is proposed with reference to
the mechanism of class C ␤-lactamases because of the high
structural similarity between the two. However, there is a lack
of sufficient experiments to verify this mechanism. Although
the nucleophile of serine in the catalytic triad is highly defini-
tive, which residue(s) actually activates the serine and the sec-
ond nucleophile of H2O to initiate deacylation are matters of
debate because of insufficient evidence.
Additionally, both Lys60 and Tyr170 are able to deprotonate
Ser57, indicating their potential as general bases. However, pro-
viding a proton to the leaving group by a general base is also
necessary for completing the catalytic reaction. Thus, the dis-
tance between the C8 hydroxyl group in monacolin J and Tyr170
In PcEST, the putative nucleophilic Ser57 is indeed neighbor-
ing the carbonyl carbon in the 2-methylbutyryl group of lovas-
tatin, satisfying the requirement of a nucleophilic reaction. Fur-
2 The abbreviations used are: PDB, Protein Data Bank; DFP, diisopropyl-fluo-
rophosphate; ITC, isothermal titration calorimetry.
1050 J. Biol. Chem. (2020) 295(4) 1047–1055

Mechanism and engineering of lovastatin esterase PcEST
Figure 4. Proposed catalytic mechanism of PcEST-mediated lovastatin hydrolysis. Two successive nucleophilic reactions initiated by the Ser57 residue in
the Ser57–Tyr170 (general base)–Lys60 (general acid) catalytic triad and a water molecule achieve efficient lovastatin hydrolysis to produce monacolin J and
2-methylbutyric acid.
(2.7 Å) or Lys60 (5 Å) determines the possibility of Tyr170 as a found within the PcEST–simvastatin complex structure (Fig.
general base, but not Lys60, in PcEST-mediated lovastatin hy- 1G). When simvastatin binds the active site, the Ser57 hydroxyl
drolysis (Fig. 2F). To further confirm this hypothesis, a series of is displaced by steric hindrance of the extra methyl in the 2Ј2-
Tyr170 or Lys60 mutants was used to assay lovastatin hydrolase dimethybutyryl side chain. This moves the Ser57 hydroxyl away
activity (Fig. 2A). All Tyr170 mutants lost lovastatin hydrolase from the carbonyl carbon of simvastatin, thus blocking nucleo-
activity. However, only Lys60 mutants with long-side-chain philic attack and unfavorable simvastatin catalysis.
substitution had abolished activity. These results indicate that
the Tyr170 phenolic hydroxyl group is an essential general base,
The hydrogen-bond network around the catalytic triad is
important for enzyme catalytic efficiency
with Lys60 acting as a general acid aiding the position and polar-
In PcEST, the Trp344 in the WGG motif and the Tyr127 at the
interface of two domains, both forming hydrogen bonds with
the catalytic triad and concurrently interacting with statins,
might be important for lovastatin hydrolysis efficiency (Fig. 2, D
and E). To verify this hypothesis, we constructed mutants that
disrupted the hydrogen-bond network at Trp344 and Tyr127. All
mutants had dramatically reduced lovastatin hydrolysis, espe-
cially W344K and Y127A inactivation mutants (Fig. 2A). These
results suggest that the hydrogen-bond network around the
catalytic triad is important for efficient lovastatin hydrolysis. A
possible explanation for W344K inactivation is that Lys344 trig-
gers a new possible catalytic triad, Ser57–Lys344–Tyr170, which
changes the correct conformation of the Ser57 hydroxyl group.
In addition, subsequent kinetic analyses of W344F and Y127F
showed that Phe replacement caused a dramatic increase in Km
and a decrease in kcat (Table 1), verifying the role of Trp344 and
Tyr127 in substrate binding and in catalysis.
ization of Tyr170
.
In terms of the deacylation mechanism, an H2O molecule
forms hydrogen bonds with both Ser57 and Tyr170 in apo PcEST
and is replaced by lovastatin upon substrate binding, making it
an ideal nucleophile to attack the acyl-enzyme intermediate to
release 2-methylbutyric acid (Fig. 2, D and E). In addition, this
H2O molecule is occupied by one DFP methyl group and simul-
taneously overlaps with the C–O bond at C8 of lovastatin in Fig.
3A, supporting the hypothesis that H2O is the second nucleo-
phile. We verified this finding using an H218O isotope-labeling
assay, which directly verified H2O as the second nucleophile.
The data showed that the m/z ratio of all characteristic 2-meth-
ylbutyric acid fragment ions containing hydroxyl groups
increased by 2, compared with a control assay with normal
H216O (Fig. 3B). To our knowledge, this is the first direct exper-
imental evidence of the nucleophilic function of H2O molecules
in the currently reported family VIII carboxylesterases.
Based on these analyses, a possible catalytic mechanism of
PcEST-mediated lovastatin hydrolysis is proposed (Fig. 4).
First, Tyr170 (general base), polarized by Lys60 (general acid), is
bonded to the Ser57 hydroxyl to activate the nucleophilic Ser57.
Next, lone pairs on the oxygen of Ser57 attack the positively
charged carbonyl carbon of lovastatin and then form an acyl-
enzyme intermediate. Moreover, Tyr170 provides a proton for
the leaving group to produce monacolin J. Subsequently, H2O is
deprotonated by Tyr170 and then nucleophilically attacks the
acyl-enzyme intermediate to release 2-methylbutyric acid.
The specific substrate-binding tunnel is also important for the
catalytic efficiency of enzymes
In PcEST, R1 (residues 222–248), R2 (residues 127–153), and
R3 (residues 290–319) regions around the active site might
define the entrance and bottom of the substrate-binding
pocket, respectively (Fig. 5). With lovastatin binding, loop 238–
245 in R1 increases the entrance size by ϳ4 Å, likely because of
a conformational change to Glu241 (Figs. 2C and 5). On the
opposite side, the Tyr127 in the ␲–␲ interaction network forms
a new hydrogen bond with Lys60 (absent in apo PcEST struc-
ture) to stabilize the general acid role of Lys60 (Fig. 2, D and E).
Analyses of reasons for the inactivity of PcEST on simvastatin
Having determined the lovastatin hydrolysis mechanism, we These changes imply that the Tyr127-containing R2 region,
further analyzed why PcEST has no activity toward simvastatin. together with loop 238–245, is a latch for the entrance of the
There are two possibilities: invalid simvastatin ligand binding substrate-binding tunnel. Our previously reported Q140L
or steric effects on nucleophilic attack. The first hypothesis was mutant in the R2 helix region, causing a decrease in Km and an
ruled out by ITC experiments, which revealed similar KD val- increase in kcat toward lovastatin (9), also indicates the impor-
ues between PcEST S57A–lovastatin and PcEST–simvastatin. tance of R2 in lovastatin hydrolysis. Further, the Phe309 benzene
These results indicate similar binding affinity to lovastatin and ring at the bottom of the substrate-binding pocket swings away
simvastatin (Fig. S2). Some evidence for the steric effect is from the hydrophobic core and forms a ␲–␲ interaction with
J. Biol. Chem. (2020) 295(4) 1047–1055 1051

Mechanism and engineering of lovastatin esterase PcEST
around the general base to regulate the catalytic state of LovD
(5). Therefore, these regions can be used as indicators to distin-
guish acyltransferase and lovastatin esterase.
Structure-guided engineering of PcEST
Our previous work showed that the main problems with
PcEST are its poor soluble expression and thermostability (9).
The highly flexible regions on protein surfaces sometimes affect
the solubility of the proteins and/or reduce their thermostabil-
ity (17, 18), because they might enhance the entropy during
protein unfolding by increasing the numbers of unfolded con-
formations (19). In the PcEST structure, the long loop in R2 and
the antiparallel ␤-sheet that may participate in the conforma-
tion stabilization of R2 helix showed high flexibility with higher
structural B factors. Considering that mutations in the R2
region are likely to significantly affect enzyme activity, we first
screened for mutation sites in the antiparallel ␤-sheet. We
found that the residues around Asp106 are almost hydrophobic
residues, involved in the stabilization of the R2 helix. However,
the residue Asp106, which is exposed on the surface and is not
bonded to any residue, is very flexible and not functionally nec-
essary (Fig. 6A). In addition, the Asp131 in R2, forming a hydro-
gen bond with Ser161, might also be involved in R2 stability and
then affect catalysis (Fig. 6B). Thus, we further engineered
Asp106 and Asp131 using Ala substitution.
Figure 5. Specific substrate-binding tunnel of PcEST. The R1, R2, and R3
regions, which might define the entrance and bottom of the substrate-bind-
ing pocket, are highlighted and marked with the corresponding residue num-
bers. The residues participating in lovastatin (yellow) binding are shown as
sticks. The three significant swings after lovastatin binding (residues Phe309
,
Glu241, and Met242) in PcEST are marked.
the lovastatin naphthalene ring. Finally, residues Tyr170, Leu306
,
Phe309, Phe310, Trp344, Gly345, Gly346, Gly347, and Leu348 ensure
the precise localization of the statin naphthalene ring. Phe54–
Ile236–His253 and Tyr127–His128–Phe129–Leu130–Met242 are
separately involved in binding the two acid side chains of lov-
astatin (Fig. 5). All of these ensure the accurate position of the
nucleophilic Ser57 hydroxyl group toward lovastatin and initi-
ate the hydrolysis reaction.
Impressively, compared with the slightly reduced catalytic
efficiency, the D106A mutant significantly improved the solu-
ble expression level. In addition, the D106A mutant presented a
Based on these analyses, a possible explanation for Y127A
inactivation is that the phenol group absent in the Y127A
mutant most likely causes a collapse of the ␲–␲ interaction
network, which reshapes the PcEST substrate entrance, result-
ing in its inactivation. However, the structure of Y127A is not
yet available to verify this hypothesis. Future studies should
investigate the Y127A inactivation mechanism.
10
nearly 1 °C increased T50 over the WT PcEST, implying an
improved thermostability. Although the catalytic efficiency and
soluble expression level of the D131A mutant were almost the
same as those of WT PcEST, the T5010 was reduced by nearly
3 °C because of mutation, indicating the importance of hydro-
gen bond for the thermostability of enzymes (Fig. 6D). The
improved solubility and thermostability of the D106A mutant
suggest promising application potential for industrial pro-
cesses, and on the other hand, the results also demonstrated the
feasibility of PcEST structure-guided enzyme engineering.
Thus, we will perform saturation mutations on other key sites,
such as the R2 loop region, to further improve the properties of
PcEST.
Comparison between the lovastatin esterase PcEST and
acyltransferase LovD
A Dali-based analysis in the PDB (16) revealed that the top
structure matching PcEST is the acyltransferase LovD from
A. terreus (PDB code 3HLB; Z score ϭ 46.8; root-mean-square
deviation ϭ 2.4 Å), which also shares the top sequence identity
(ϳ40%) with PcEST among function-identified proteins. LovD
mainly catalyzes lovastatin synthesis and concurrently has
weak lovastatin hydrolase activity. Ser76–Tyr188–Lys79 was
reported as its catalytic triad (4). The highly similar primary
sequence and three-dimensional core structure between acyl-
transferase LovD and lovastatin esterase PcEST increases the
difficulty of subsequent screening for new efficient lovastatin
hydrolases (Figs. S1 and S3). Therefore, the ability to quickly
distinguish the acyltransferases and lovastatin esterases will
provide notable convenience for screening new specific lovas-
tatin esterases.
Conclusion
The first high-resolution structures of a specific lovastatin
esterase were reported in this work. Structure-based biochem-
ical analyses and mutagenesis studies revealed the specific and
efficient lovastatin hydrolysis mechanism and the structure–
function relationships of PcEST, which will guide subsequent
lovastatin esterase screening and engineering. Furthermore, we
generated a variant with improved solubility and thermostabil-
ity, D106A, thus indicating the potential to improve the indus-
trial application of PcEST through structure-guided enzyme
engineering.
In LovD, a relatively wider entrance between R1 and R2 is
thought to satisfy the ACP-dependent synthesis (Fig. S3) (4).
Moreover, the key WGG motif involving the hydrogen-bond
network of PcEST active site is replaced by FGG in all reported
LovD homologous acyltransferases (Fig. S1). Additionally, the
Phe309–Phe310 necessary for lovastatin localization in PcEST is
displaced by the Ile325–Tyr327 in LovD (Fig. S3), whose confor-
Materials and methods
Gene cloning and mutagenesis
The PCR-amplified PcEST fragment from a PcEST–pEASY–
mation changes to generate a new hydrogen-bond network Blunt E2 plasmid template (2) was ligated with the pEASY-E1
1052 J. Biol. Chem. (2020) 295(4) 1047–1055

Mechanism and engineering of lovastatin esterase PcEST
Figure6. Structure-guidedenzymeengineeringofPcEST. AandB, zoomedviewofthelocalenvironmentofAsp106 (A)andAsp131(B)inthePcESTstructure.
The R2 region of PcEST is colored blue. The related residues are shown with sticks, and the hydrogen bond is marked by a black dashed line. C and D, expression
(C) and thermostability (D) of PcEST and its mutants. The soluble (Sup) and insoluble portions (Pre) of PcEST expressed in BL21 (DE3). Thermostability T5010 is
defined as the temperature where 50% of initial enzyme activity (green line) is lost following 10-min heat treatment. Each point represents the means from two
independent replicates with standard deviation.
vector (Transgen Biotech) to construct the pEASY–E1–PcEST A and then purified with nickel–nitrilotriacetic acid resin to
recombinant plasmid, which expresses PcEST with an N-ter- remove the His6–smt3 fusion tag. Finally, the target protein was
minal His6 tag. The PcEST–pET28a–smt3 plasmid was con- further purified using a Superdex75 10/300 GL column (GE
structed using NdeI and XhoI restriction enzymes and ex- Healthcare) and eluted with buffer A. Protein fractions from
presses PcEST protein with an Ulp1-cleavable N-terminal each purification step were analyzed by SDS-PAGE. All target
His6–smt3 fusion tag. All PcEST mutants used in this study proteins were concentrated to 16 mg/ml in buffer A for subse-
were created using the QuikChange site-directed mutagenesis quent protein screening and enzymatic activity.
method, with the PcEST–pET28a–smt3 plasmid as the tem-
Crystallization, data collection, and structure determination
plate. All primers are listed in Table S1. Recombinant plasmids
Crystallization conditions were screened in 48-well plates at
18 °C by the sitting-drop vapor-diffusion method. A 1:1 protein:
reservoir solution ratio and 2-␮l total drop size was used for
were confirmed by DNA sequencing.
Protein expression and purification
All PcEST proteins were expressed in Escherichia coli crystallization. Apo PcEST crystals, containing a 17 residue
BL21(DE3) (Transgen Biotech) as described previously (2). N-terminal flexible extension, were grown at 10 mg/ml in 4 M
After induction, the cells were harvested by centrifugation and sodium formate. However, well-ordered enzyme–ligand com-
then sonicated in lysis buffer (20 mM Tris-HCl, pH 8.0, 300 mM plex crystals could not be obtained from this procedure. We
NaCl, 10 mM imidazole, 5 mM ␤-mercaptoethanol, and 10 then removed the flexible extension using the vector PcEST–
␮g/ml DNase I). The resulting extract was first purified by pET28a–smt3 in subsequent crystallization experiments.
nickel–nitrilotriacetic acid–agarose resin (Qiagen). His6-
For the enzyme–ligand complex, 10 mg/ml PcEST (WT or
tagged proteins were eluted with buffer containing 20 mM Tris- the inactivated mutant S57A) was incubated with 3.5 mM
HCl, pH 8.0, 300 mM NaCl, 250 mM imidazole, and 1 mM ligand, monacolin J, simvastatin, or lovastatin at 4 °C for 2 h.
␤-mercaptoethanol. Next, PcEST–His6 protein was loaded Diffraction-quality crystals of S57A-lovastatin were obtained in
onto a HiLoad 16/60 Superdex 200 column (GE Healthcare) 2–3 days when grown in 200 mM calcium acetate, 100 mM Tris-
with buffer A containing 10 mM Tris-HCl, pH 8.0, and 100 mM HCl, pH 7.0, and 20% (w/v) PEG 3000. The PcEST–monacolin
NaCl. His6–smt3 fusion PcEST (WT and mutants) proteins J and PcEST–simvastatin crystals were grown in crystallization
were dialyzed and digested with Ulp1 overnight at 4 °C in buffer buffer (200 mM calcium acetate, 100 mM sodium cacodylate
J. Biol. Chem. (2020) 295(4) 1047–1055 1053

Mechanism and engineering of lovastatin esterase PcEST
trihydrate, pH 6.5, and 18% (w/v) PEG 8000) for 2 days. The After centrifugation at 4 °C, the supernatant and resuspended
crystals were harvested and cryoprotected in crystallization precipitate fractions were analyzed by SDS-PAGE.
solution containing 16% glycerol or 20% (v/v) PEG 400 and
Isothermal titration calorimetry assays
flash-cooled in liquid nitrogen.
The diffraction data were collected at the National Center for
ITC experiments were performed at 25 °C with a MicroCal
Protein Sciences Shanghai (Shanghai, China), using Beamline iTC200 (GE Healthcare) using 60 ␮M enzyme in the sample cell
BL19U1 (apo PcEST), or the Beijing Synchrotron Radiation and 1 mM ligand in the syringe. Thirteen injections (3 ␮l each)
Facility (Beijing, China) for PcEST–monacolin J, PcEST– were used. The dilution heat for each ligand was measured in a
simvastatin, and S57A–lovastatin complex structures. The data separate titration experiment by titrating ligand into buffer.
were processed using HKL3000 or HKL2000 software (20, 21). Consecutive injections were separated by at least 2 min. The
The apo PcEST structure was solved with molecular replace- ITC data were analyzed with one sites model using Origin 8.0
ment using Phaser (22) and coordinates of LovD6 from A. ter- software (OriginLab).
reus as a search model (PDB code 4LCL). LovD6 shares 36%
H218O isotope labeling assays
identity over 147 residues with PcEST. Two protein molecules
The H218O used in the reaction buffer was purchased from
Macklin (CAS: 14314-42-2). To obtain sufficient 2-methylbu-
tyric acid for GC-MS detection, 10 mM lovastatin was com-
pletely hydrolyzed by 0.05 ␮M PcEST at 30 °C and 110 rpm
overnight. The reaction mixture (1 ␮l) was diluted by 100%
anhydrous methanol and analyzed by GC-MS using an Agilent
7890A-5975C system equipped with a HP-5 column (30 m ϫ
250 ␮m ϫ 0.25 ␮m). Helium (constant flow 1 ml/min) was used
as the carrier gas. The injector temperature was 250 °C, and the
following temperature program was applied: initial tempera-
ture 40 °C for 0 min, 10 °C/min to 250 °C in the ramp step, and
maintained at 250 °C for 10 min. A 2-methylbutyric acid stan-
dard was used as a control.
were found in the asymmetric unit. The phases for subsequent
enzyme–ligand complex structures were also obtained by
molecular replacement using apo PcEST as the search model.
An additional electron density appeared at the proposed sub-
strate-binding pocket. The structures were refined using Ref-
mac or Phenix.refine software and were manually corrected in
Coot in iterative rounds (23–25). Final model quality was
checked using MolProbity (26). A data collection and final
refinement statistics summary is found in Table S2. PyMOL
was used to prepare all structural figures (27).
Enzymatic characterization of PcEST and its mutants in vitro
The general reaction system consisted of 0.05 ␮M WT PcEST
protein or 5 ␮M mutants, 400 ␮M lovastatin and 50 mM Tris-
HCl pH 8.0 buffer. After incubation at 30 °C for 30 min, the
hydrolysis reaction was quenched by adding an equal volume of
100% anhydrous methanol. The HPLC detection method was
described previously (2).
Author contributions—Y. L. data curation; Y. L. and X. L. formal
analysis; Y. L. and X. L. funding acquisition; Y. L. and X. L. valida-
tion; Y. L. investigation; Y. L. visualization; Y. L. methodology; Y. L.
writing-original draft; Y. L. and X. L. project administration; X. L.
conceptualization; X. L. supervision; X. L. writing-review and
editing.
For the determination of thermostability, the purified pro-
teins (0.5 mg/ml) were preincubated at temperatures from 25 to
50 °C for 10 min using the temperature gradient function on
PCR (Bio-Rad T100*) and then quickly cooled on ice. The lov-
astatin hydrolysis reaction was performed as described above.
We assessed kinetic parameters (Km and kcat) of PcEST,
Y127F, W344F, D106A, and D131A mutants at 30 °C. First,
enzyme and lovastatin substrate concentrations and reaction
times were optimized. The final lovastatin concentration varied
from 3 to 200 ␮M for PcEST (0.01 ␮M), D106A (0.005 ␮M), and
D131A (0.01 ␮M); 25–700 ␮M for Y127F (0.5 ␮M); and 15–700
␮M for W344F (3 ␮M) in 50 mM Tris-HCl pH 8.0 buffer. At least
two independent experiments were performed for each reac-
tion time point. The kinetic curve was drawn by fitting the
initial rate data into the Michaelis–Menten equation using
GraphPad Prism version 5.0.0 software.
Acknowledgments—We thank Dr. Zengqiang Gao and the staffs of the
3W1B Beamline at the Beijing Synchrotron Radiation Facility and
the staffs of the BL19U1 Beamline at National Center for Protein
Sciences Shanghai for assistance during data collection. Cong Wang
from the Qingdao Institute of Bioenergy and Bioprocess Technology of
the Chinese Academy of Sciences provided technical support for MS.
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