STRUCTURE O FUNCTION O BIOINFORMATICS
Crystal structure of a triacylglycerol lipase
from Penicillium expansum at 1.3 A ˚ determined
by sulfur SAD
Chuanbing Bian,1Cai Yuan,1Liqing Chen,2Edward J. Meehan,2Longguang Jiang,1
Zixiang Huang,1Lin Lin,3and Mingdong Huang1*
1State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian, 350002, China
2Laboratory for Structural Biology, Department of Chemistry, University of Alabama in Huntsville, Huntsville, Alabama 35899
3Department of Biology, Fujian Normal University, Fuzhou 350007, China
Key words: Penicillium expansum; lipase; X-ray crystallography; sulfur SAD.
Triacylglycerol lipases (EC 18.104.22.168) are present in many
microbes. Lipases catalyze the hydrolysis of long-chain
triglycerides into fatty acids and glycerol at the interface
between the water insoluble substrate and the aqueous
phase. Lipases can also catalyze the reverse esterification
reaction to form glycerides under certain conditions.
Lipases of microbial origin are of considerable commer-
cial interest for wide variety of biotechnological applica-
tions in industries, including detergent, food, cosmetic,
pharmaceutical, fine chemicals, and biodiesel.1–3Nowa-
days, microbial lipases have become one of the most
important industrial enzymes.
PEL (Penicillium expansum lipase) is a fungal lipase
from Penicillium expansum strain PF898 isolated from Chi-
nese soil that has been subjected to several generations of
mutagenesis to increase its enzymatic activity.4,5PEL
belongs to the triacylglycerol lipases family, and its catalytic
characteristics have been studied.6,7The enzyme has been
used in Chinese laundry detergent industry for several
years (http://www.leveking.com). However, the poor ther-
mal stability of the enzyme limits its application. To fur-
ther study and improve this enzyme, PEL was cloned and
sequenced.8–10Furthermore, it was overexpressed in
Pichia pastoris.6PEL contains GHSLG sequence, which is
the lipase consensus sequence Gly-X1-Ser-X2-Gly,11but
has a low amino acid sequence identities to other lipases
(see Supporting Information Figure S1). The most similar
lipases are Rhizomucor miehei (PML12) and Rhizopus
niveus (PNL13) with a 21% and 20% sequence identities to
PEL, respectively. Interestingly, the similarity of PEL with
the known esterases is somewhat higher with 24%
sequence identity to feruloyl esterase A.14
Here, we report the 1.3 A˚resolution crystal structure
of PEL determined by sulfur SAD phasing. This structure
not only presents a new lipase structure at high resolu-
tion, but also provides a structural platform to analyze
the published mutagenesis results. The structure may also
open up new avenues for future protein engineering
study on PEL.
Additional Supporting Information may be found in the online version of this
Grant sponsor: Fujian Province; Grant number: 2007F30105; Grant sponsor: Nat-
ural Science Foundation of China; Grant numbers: 30811130467, 30625011; Grant
sponsor: Ministry of Science of Technology; Grant numbers: 2006AA02A313,
2007CB914304; Grant sponsor: Chinese Academy of Sciences; Grant number:
KSCX2-YW-R-082; Grant sponsor: US Department of Energy, Office of Science,
Office of Basic Energy Sciences; Grant number: W-31-109-Eng-38; Grant sponsor:
*Correspondence to: Mingdong Huang, State Key Laboratory of Structural
Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese
Academy of Sciences, 155 Yang Qiao West Road, Fuzhou, Fujian, 350002, China.
Received 15 September 2009; Revised 5 December 2009; Accepted 7 December
Published online 21 December 2009 in Wiley InterScience (www.interscience.wiley.
com). DOI: 10.1002/prot.22676
C 2009 WILEY-LISS, INC.
Purification and crystallization
The recombinant PEL was expressed in Pichia pastoris
cells and purified to homogeneity.6,15Crystals suitable
for X-ray diffraction analysis were obtained by the sit-
ting-drop vapor diffusion method with ammonia sulfate
as the precipitant at 298 K. No inhibitor was included
in the crystallization setup. The detailed purification
and crystallization procedures were described else-
where.15For data collection, the crystals were soaked
briefly in the mother liquor containing 15% ethylene
glycol, and then flash-cooled to 100 K in a cold nitrogen
stream. The crystals diffracted to a resolution of 2.3 A˚
on an in-house rotating-anode generator, but to much
higher resolution (1.3 A˚) with the APS beam line
SER-CAT 22ID at a wavelength of 1.00 A˚. For phasing,
method (S-SAD) data set was collected at 1.9 A˚to a re-
solution of 2.0 A˚. The synchrotron data were processed
and scaled with HKL200016(see the data collection
statistics in Table I).
Structure determination and refinement
The structure was solved by S-SAD data of PEL at
2.0 A˚. This S-SAD data set was collected to high redun-
dancy (20.2) and high signal/noise ratio (50.5). All
18 sulfur atoms in the asymmetric unit were located
using the program SHELXD.17,18The initial phases were
calculated from these sulfur atom positions by program
SOLVE and RESOLVE,19–21giving a figure-of-merit of
0.47 and 0.14 for acentric and centric reflections, respec-
tively, and yielding an interpretable electron density map.
Model rebuilding was carried out by ARP/wARP against
a native data set at 1.3 A˚(Supporting Information Figure
S2). The structure was finally refined and rebuilt using
REFMAC22–24and COOT,25gave an R factor of 18.7%
and a free R factor of 20.9%. The model was analyzed
using PyMol.26A total of 429 water molecules were
included in the final model. The final PEL model has
only three residues in the disallowed region of the Rama-
chandran plot, as shown by PROCHECK.27
The final PEL model contains two molecules (named
A and B) in the asymmetric unit. These two molecules
are similar to each other with a root mean square (RMS)
deviations of 0.24 A˚when all Ca atoms were compared.
The average temperature factors value for all atoms of
the two molecules are 13.19 and 12.5 A˚2, respectively.
The lid domain in molecule B (residues The68-Asp74)
was clearly visible in the electron density maps, and
formed a helix turn. However, the corresponding residues
in the molecule A were not seen in electron density
maps, and were thus omitted out from the final model.
RESULTS AND DISCUSSION
The 1.3 A˚structure of PEL shows a globular and com-
pact single-domain protein with a dimension of 46 3 41
3 40 A˚(Fig. 1). PEL has a modified a/b hydrolase fold
that consists of seven b-strands in a parallel b-sheet, sur-
rounded by four long a-helices and six small a-helices
with only three or four amino acid residues. There are
three a-helices on one side of the b-sheet and seven on
the other side. Compared with the canonical a/b hydro-
lase fold,28PEL has an additional a-helix (a1) at the
N-terminus and two short helices (a9 and a10) at
the C-terminus, but lacks the last b-strand at the
Active site architecture
The active site of PEL was identified to be S132, D188,
and H241. The spatial arrangement of this catalytic triad
superimposes quite well to the closest enzymes, feruloyl
esterase A,14or Rhizomucor miehei lipase (PDB code:
3TGL29), with the RMS deviation for all atoms less
than 0.2 A˚. S132 is the nucleophile that needs to be
Data Collection and Refinement Statistics of PEL Crystal
Data setNative data set S-SAD data set
X-ray wavelength (?)
Resolution range (?)
Unit cell parameters (?)
a 5 b 5 88.27,
c 5 126.62
a 5 b 5 88.20,
c 5 126.53
Solvent content (%)
Resolution range (?)
No. working/test set
No. atoms total
Root mean square bonds (?)
Root mean square angles (8)
Most favorable residues
Average B-factors (?2)
Values in parentheses correspond to the highest resolution shells.
aRsym 5 ShklSj|I(hkl;j) 2 <I(hkl)>|/(ShklSj<I(hkl)>), where I(hkl;j) is the jth
measurement of the intensity of the unique reflection (hkl) and I(hkl) is the mean
overall symmetry related measurements.
bRfreewas calculated from a randomly chosen 5% of unique reflections.
C. Bian et al.
activated by the histidine and aspartic acid residues in a
mechanism common to all serine hydrolases, and attacks
the substrate to form a covalent acyl-enzyme intermedi-
ate. All serine hydrolases contain an oxyanion hole, gen-
erally made up of two main-chain amide hydrogen
atoms, that stabilizes the tetrahedral intermediates that
occur during both the acylation and the deacylation
steps.30In the current PEL structure, two amide groups
in residues 130 and 131 were identified to be the oxyan-
ion hole based on the structural comparison to Rhizomu-
cor miehei lipase in the presence of inhibitor.29
Lipases are characterized by their drastically increased
activity when acting at the lipid-water interface of micel-
lar, a phenomenon called interfacial activation.31This
property is a result of the presence of a lid domain that
covers the active site. Lid domain is a short helical seg-
ment or a surface loop, and is often amphipathic in
chemical nature. On exposing to the lipid phase in the
lipid–water interface, this amphipathic lid undergoes
conformational change, moves away from the active site
serine, thus turning the ‘‘closed" form of the enzyme into
an ‘‘open’’ form, with the active site now accessible to
the solvent. At the same time, a large hydrophobic sur-
face in lipase is exposed, facilitating the binding of the
lipase to the substrates.32In the current PEL structure,
(sequences I68, T69, D70, F71, V72, N73, D74, I75, and
D76). All the hydrophobic residues of the lid domain
point toward the active site cleft (see Fig. 2). Interest-
ingly, these hydrophobic residues do not have many con-
tacts with the hydrophobic residues in the active site
cleft, leaving cavities between the catalytic S132 and the
lid domain. Moreover, the lid domain has relatively high-
temperature factor (18.6 A˚2compared with 12.5 A˚2of all
atoms in the molecule), and form a distorted helix. The
lid domain in the molecule A of the crystal was com-
pletely disordered. These facts, taken together, suggest
that the lid domain of PEL is quite flexible.
Most lipases act at a specific position on the glycerol
backbone of lipid substrate. However, PEL does not show
preference on acyl position of the glycerol backbone of
lipid substrates,33and converts triacylglycerol substrates
to glycerol and free fatty acids. PEL also shows broad
Overall structure of PEL. Active site residues are represented in sticks.
The lid domain (I68-D76) structure of PEL in the molecule (B).
The hydrophobic residues of the lid domain have only a
few contacts with the active site cleft and thus may be flexible.
The corresponding region in another molecule (A) of the asymmetric
unit was disordered and not included in the structure model. The
stick radii of the lid domain residues are proportional to their contact
areas to the rest of PEL.
Structure of a Lipase at 1.3 A˚
substrate specificity: it hydrolyzes lipids with either long
or short fatty acid chains. A series of PEL mutants
(K55R, E83V, D92P, P163A, R182K, and K202A) were
identified through random mutation method to increase
PEL thermostability and to either lower or raise its opti-
mal hydrolysis temperature for the application as an
addition for laundry detergents.34,35Our current high-
resolution PEL model provides a structural platform to
analyze the structure–function relationship of these PEL
Mutations of P163A, R182K, and K202A take place
near the active site, and may thus perturb the active site
conformation. There are quite a few cavities inside the
PEL structure. Residues P163, R182, and K202 are close
to one of the largest cavities near to the catalytic triad.
P163A mutant demonstrated a significant effect on PEL
optimal operating temperature (158C lower than wild
type PEL). The current PEL structure shows that P163 is
close to the active site and to a largest cavity close to the
catalytic triad (9 A˚from P163 to active site S132). The
replacement of a proline residue by alanine residue can
increase the conformational flexibility of PEL active site,
and thus lower the optimal hydrolysis temperature of the
mutant. Further structural studies on the PEL mutants
or its complex with inhibitors are needed to further our
understanding on the structure–function relationship of
The authors thank Dr. Lirong Chen and Prof. Bi-
Cheng Wang of The University of Georgia for help with
S-SAD data collection and the staffs of the APS SER-CAT
beamline 22ID for help during our data collection. The
atomic coordinates and structure factors of PEL have
been depositedin theprotein
www.rcsb.org) as 3G7N.
1. Jaeger KE, Dijkstra BW, Reetz MT. Bacterial biocatalysts: molecular
biology, three-dimensional structures, and biotechnological applica-
tions of lipases. Annu Rev Microbiol 1999;53:315–351.
2. Kim Hyung K. Molecular structures and catalytic mechanism of
bacterial lipases. Korean J Microbiol Biotechnol 2003;31:311–321.
3. Schmid RD, Verger R. Lipases: interfacial enzymes with attractive
applications. Angew Chem-Int Ed 1998;37:1609–1633.
4. Shi Q. Studies on alkaline lipase. I. Screening and purification of
the microorganisms. Acta Microbiol Sin 1981;8:109–110.
5. Shi Q. Studies on alkaline lipase. II. Mutagenesis and breed of the
microorganisms. Acta Microbiol Sin 1982;9:162–164.
6. Yuan C, Lin L, Shi Q, Wu S. Overexpression of Penicillium expansum
lipase gene in Pichia pastoris. Chin J Biotechnol 2003;19:231–235.
7. Zheng Y, Shi Q, Wu S. Effects of surfactants on alkaline lipase. Ind
8. Lin L, Xie B, Yang G, Shi Q. Cloning and sequenceing of genomic
DNA encoding alkaline lipase from Penicillium expansum PF898.
Chin J Biochem Mol Biol 2003;19:12–16.
9. Lin L, Xie B, Yang G, Shi Q, Lin Q, Xie L, Wu X, Wu S. Clon-
ing and sequence analysis of cDNA encoding alkaline lipase from
Penicillium expansum PF898. Chin J Biochem Mol Biol 2002;18:
10. Lin L, Shi Q, Guo X, Wu S, Wu X, Xie L. Purification and
N-terminal sequencing of an alkaline lipase from Penicillium expan-
sum. J Xiamen Univ (Nat Sci) 2002;41:600–604.
11. Persson B, Bengtssonolivecrona G, Enerback S, Olivecrona T, Jorn-
vall H. Structural features of lipoprotein-lipase—lipase family rela-
tionships, binding interactions, non-equivalence of lipase cofactors,
vitellogenin similarities and functional subdivision of lipoprotein-
lipase. Eur J Biochem 1989;179:39–45.
12. Derewenda U, Brzozowski AM, Lawson DM, Derewenda ZS. Catal-
ysis at the interface: the anatomy of a conformational change in a
triglyceride lipase. Biochemistry 1992;31:1532–1541.
13. Kohno M, Funatsu J, Mikami B, Kugimiya W, Matsuo T, Morita Y.
The crystal structure of lipase II from Rhizopus niveus at 2.2 ang-
strom resolution. J Biochem 1996;120:505–510.
14. Benoit I, Asther M, Sulzenbacher G, Record E, Marmuse L, Parsie-
gla G, Gimbert I, Bignon C. Respective importance of protein
folding and glycosylation in the thermal stability of recombinant
feruloyl esterase A. Febs Lett 2006;580:5815–5821.
15. Bian C, Yuan C, Lin L, Lin J, Shi X, Ye X, Huang Z, Huang M.
Purification and preliminary crystallographic analysis of a Penicil-
lium expansum lipase. Biochim Biophys Acta 2005;1752:99–102.
16. Otwinowski Z, Minor W. Processing of X-ray diffraction data col-
lected in oscillation mode. In: Carter CW, Jr, editor. Methods in en-
zymology, Vol. 276. New York: Academic Press; 1997. pp 307–326.
17. Schneider TR, Sheldrick GM. Substructure solution with SHELXD.
Acta Crystallogr D Biol Crystallogr 2002;58 (Part 10, Part 2):1772–
18. Nanao MH, Sheldrick GM, Ravelli RB. Improving radiation-damage
substructures for RIP. Acta Crystallogr D Biol Crystallogr 2005;61
19. Terwilliger TC. SOLVE and RESOLVE: automated structure solution
and density modification. Methods Enzymol 2003;374:22–37.
20. Terwilliger T. SOLVE and RESOLVE: automated structure solution,
density modification and model building. J Synchrotron Radiat
2004;11 (Part 1):49–52.
21. Wang JW, Chen JR, Gu YX, Zheng CD, Jiang F, Fan HF, Terwilliger
TC, Hao Q. SAD phasing by combination of direct methods with
the SOLVE/RESOLVE procedure. Acta Crystallogr D Biol Crystal-
logr 2004;60 (Part 7):1244–1253.
22. Murshudov GN, Vagin AA, Dodson EJ. Refinement of macromolec-
ular structures by the maximum-likelihood method. Acta Crystal-
logr D Biol Crystallogr 1997;53 (Part 3):240–255.
23. Winn MD, Isupov MN, Murshudov GN. Use of TLS parameters to
model anisotropic displacements in macromolecular refinement.
Acta Crystallogr D Biol Crystallogr 2001;57 (Part 1):122–133.
24. Winn MD, Murshudov GN, Papiz MZ. Macromolecular TLS refine-
ment in REFMAC at moderate resolutions. Methods Enzymol 2003;
25. Emsley P, Cowtan K. Coot: model-building tools for molecular
graphics. Acta Crystallogr D Biol Crystallogr 2004;60 (Part 12, Part
26. DeLano WL. The PyMol molecular graphics system. San Carlos,
CA: DeLano Scientific; 2004.
27. Laskowski RA, MacArthur MW, Mass DS, Thornton JM. PRO-
CHECK: a program to check the stereochemical quality of protein
structures. J Appl Crystallogr 1993;26:283–291.
28. Ollis DL, Cheah E, Cygler M, Dijkstra B, Frolow F, Franken SM,
Harel M, Remington SJ, Silman I, Schrag J, Sussman JL, Verschue-
ren KHG, Goldman A. The a/b hydrolase fold. Protein Eng 1992;5:
29. Derewenda ZS, Derewenda U, Dodson GG. The crystal and molecu-
lar structure of the Rhizomucor miehei triacylglyceride lipase at 1.9
A resolution. J Mol Biol 1992;227:818–839.
C. Bian et al.
30. Kraut J. Serine proteases—structure and mechanism of catalysis. Download full-text
Annu Rev Biochem 1977;46:331–358.
31. Turner NA, Needs EC, Khan JA, Vulfson EN. Analysis of conforma-
tional states of Candida rugosa lipase in solution: implications for
mechanism of interfacial activation and separation of open and
closed forms. Biotechnol Bioeng 2001;72:108–118.
32. Jaeger KE, Reetz MT. Microbial lipases form versatile tools for bio-
technology. Trends Biotechnol 1998;16:396–403.
33. Zheng Y, Gong F, Shi Q, Wu S. Studies on the catalytic characters of
lipase from Pencillium expansum. Pharm Biotechnol 2000;7:98–101.
34. Zheng Y, Shi Q, Wu S. R&D of new type enzyme in detergent: alka-
line lipase. Fine Specialty Chem 2002;10:21–24.
35. Zheng Y, Wu S, Shi Q. A review on the alkaline lipase of microor-
ganism used for detergent. China Surf Detergent Cosmet 2001;31:35–39.
36. Yuan C. The expression of Penicillium expansum lipase gene and its
molecular mutants. Fuzhou: Fujian Normal University; 2003. 74 p.
Structure of a Lipase at 1.3 A˚