Crystal structure of a triacylglycerol lipase from Penicillium expansum at 1.3 Å determined by sulfur SAD

This is the authors' abstract. We don't add key points for this paper.

Triacylglycerol lipases (EC 3.1.1.3) are present in many different organisms including animals, plants, and 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 commercial interest for wide variety of biotechnological applications in industries, including detergent, food, cosmetic, pharmaceutical, fine chemicals, and biodiesel.1-3 Nowadays, 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 Chinese soil that has been subjected to several generations of mutagenesis to increase its enzymatic activity.4, 5 PEL belongs to the triacylglycerol lipases family, and its catalytic characteristics have been studied.6, 7 The enzyme has been used in Chinese laundry detergent industry for several years (http://www.leveking.com). However, the poor thermal stability of the enzyme limits its application. To further study and improve this enzyme, PEL was cloned and sequenced.8-10 Furthermore, it was overexpressed in Pichia pastoris.6 PEL contains GHSLG sequence, which is the lipase consensus sequence Gly-X1-Ser-X2-Gly,11 but 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 Å resolution crystal structure of PEL determined by sulfur SAD phasing. This structure not only presents a new lipase structure at high resolution, 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. The recombinant PEL was expressed in Pichia pastoris cells and purified to homogeneity.6, 15 Crystals suitable for X-ray diffraction analysis were obtained by the sitting-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 elsewhere.15 For 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 Å on an in-house rotating-anode generator, but to much higher resolution (1.3 Å) with the APS beam line SER-CAT 22ID at a wavelength of 1.00 Å. For phasing, a sulfur single wavelength anomalous diffraction method (S-SAD) data set was collected at 1.9 Å to a resolution of 2.0 Å. The synchrotron data were processed and scaled with HKL200016 (see the data collection statistics in Table I). The structure was solved by S-SAD data of PEL at 2.0 Å. This S-SAD data set was collected to high redundancy (20.2) and high signal/noise ratio (50.5). All 18sulfur atoms in the asymmetric unit were located using the program SHELXD.17, 18 The initial phases were calculated from these sulfur atom positions by program SOLVE and RESOLVE,19-21 giving a figure-of-merit of 0.47 and 0.14 for acentric and centric reflections, respectively, and yielding an interpretable electron density map. Model rebuilding was carried out by ARP/wARP against a native data set at 1.3 Å (Supporting Information Figure S2). The structure was finally refined and rebuilt using REFMAC22-24 and COOT,25 gave an R factor of 18.7% and a free R factor of 20.9%. The model was analyzed using PyMol.26 A 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 Ramachandran 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 Å when all Cα atoms were compared. The average temperature factors value for all atoms of the two molecules are 13.19 and 12.5 Å2, 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. The 1.3 Å structure of PEL shows a globular and compact single-domain protein with a dimension of 46 × 41 × 40 Å (Fig. 1). PEL has a modified α/β hydrolase fold that consists of seven β-strands in a parallel β-sheet, surrounded by four long α-helices and six small α-helices with only three or four amino acid residues. There are three α-helices on one side of the β-sheet and seven on the other side. Compared with the canonical α/β hydrolase fold,28 PEL has an additional α-helix (α1) at the N-terminus and two short helices (α9 and α10) at the C-terminus, but lacks the last β-strand at the C-terminus. Overall structure of PEL. Active site residues are represented in sticks. 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,14 or Rhizomucor miehei lipase (PDB code: 3TGL29), with the RMS deviation for all atoms less than 0.2 Å. S132 is the nucleophile that needs to be 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 intermediate. All serine hydrolases contain an oxyanion hole, generally made up of two main-chain amide hydrogen atoms, that stabilizes the tetrahedral intermediates that occur during both the acylation and the deacylation steps.30 In the current PEL structure, two amide groups in residues 130 and 131 were identified to be the oxyanion hole based on the structural comparison to Rhizomucor miehei lipase in the presence of inhibitor.29 Lipases are characterized by their drastically increased activity when acting at the lipid-water interface of micellar, a phenomenon called interfacial activation.31 This property is a result of the presence of a lid domain that covers the active site. Lid domain is a short helical segment 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 surface in lipase is exposed, facilitating the binding of the lipase to the substrates.32 In the current PEL structure, the lid domain was identified as residues 68–76 (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). Interestingly, these hydrophobic residues do not have many contacts 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 Å2 compared with 12.5 Å2 of all atoms in the molecule), and form a distorted helix. The lid domain in the molecule A of the crystal was completely disordered. These facts, taken together, suggest that the lid domain of PEL is quite flexible. 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. 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,33 and converts triacylglycerol substrates to glycerol and free fatty acids. PEL also shows broad 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 optimal hydrolysis temperature for the application as an addition for laundry detergents.34, 35 Our current high-resolution PEL model provides a structural platform to analyze the structure–function relationship of these PEL mutants. 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 (15°C 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 Å 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 this lipase. 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 deposited in the protein data bank (http://www.rcsb.org) as 3G7N. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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