Kinetic and structural characterization of triacylglycerol lipases possessing phospholipase A1 activity

The pancreatic lipase gene family displays various substrate selectivities for triglycerides and phospholipids. The structural basis for this difference in substrate specificity has not been definitively established. Based on a kinetic comparative study between various pancreatic lipase family members, we showed here that porcine pancreatic lipase (PPL), which was so far classified as “classical lipase”, was able to hydrolyze phosphatidylcholine (PC). Amino acid sequence alignments revealed that Val260 residue in PPL lid could be critical for the interaction with lipid substrate. Molecular dynamics was applied to investigate PC binding modes within the catalytic cavity of PPL and human pancreatic lipase (HPL), aiming to explain the difference of specificity of these enzymes towards phospholipids. Results showed that with HPL, the oxyanion hole was not able to accommodate the PC molecule, suggesting that no activity could be obtained. With PPL, the formation of a large pocket involving Val260 allowed the PC molecule to come near the catalytic residues, suggesting that it could be hydrolyzed. One more interesting finding is that human pancreatic lipase related protein 2 could hydrolyze phospholipids through its PLA₁ and PLA₂ activities. Overall, our study shed the light on new structural features of the phospholipase activity of pancreatic lipase family members.     

Cloning and characterization of the acid lipase from castor beans

Castor bean endosperm contains a well known acid lipase activity that is associated with the oil body membrane. In order to identify this enzyme, proteomic analysis was performed on purified oil bodies. A approximately 60-kDa protein was identified (RcOBL1), which shares homology with a lipase from the filamentous fungus Rhizomucor miehei. RcOBL1 contains features that are characteristic of an alpha/beta-hydrolase, such as a putative catalytic triad (SDH) and a conserved pentapeptide (GXSXG) surrounding the nucleophilic serine residue. RcOBL1 was expressed heterologously in Escherichia coli and shown to hydrolyze triolein at an acid pH (optima approximately 4.5). RcOBL1 can hydrolyze a range of triacylglycerols but is not active on phospholipids. The activity is sensitive to the serine reagent diethyl p-nitrophenyl phosphate, indicating that RcOBL1 is a serine esterase. Antibodies raised against RcOBL1 were used to show that the protein is restricted to the endosperm where it is associated with the surface of oil bodies. This is the first evidence for the molecular identity of an oil body-associated lipase from plants. Sequence comparisons reveal that families of OBL1-like proteins are present in many species, and it is likely that they play an important role in regulating lipolysis.     

Catalytic residues of group VIB calcium-independent phospholipase A2 (iPLA2gamma)

Although human group VIB calcium-independent phospholipase A(2) (iPLA(2)gamma) contains the lipase-consensus sequence Gly-Xaa-Ser-Xaa-Gly in the C-terminal half, its overall sequence exhibits a week similarity to those of other PLA(2)s, and thus no information on the catalytic site has been available. Here we show that the C-terminal region of human iPLA(2)gamma is responsible for the enzymatic activity. Comparison of this catalytic domain with those of the mouse homologue, human cytosolic PLA(2) (cPLA(2)), and the plant PLA(2) patatin reveals that an amino acid sequence of a short segment around Asp-627 of iPLA(2)gamma is conserved among these PLA(2)s, in addition to the Ser-483-containing lipase motif; the corresponding serine and aspartate in cPLA(2) and patatin are known to form a catalytic dyad. Since substitution of alanine for either Ser-483 or Asp-627 results in a loss of the PLA(2) activity, we propose that Ser-483 and Asp-627 of human iPLA(2)gamma constitute an active site similar to the Ser-Asp dyad in cPLA(2) and patatin.     

Identification of novel phospholipase A2 group IX members in metazoans

Sequence homologues of the bacterium Streptomyces violaceoruber and sea anemone Nematostella vectensis PLA₂ pfam09056 members were identified in several bacteria, fungi and metazoans illustrating the evolution of this PLA₂ sub-family. Comparison of their molecular structures revealed that bacteria and fungi members are part of the GXIV of PLA₂s while metazoan representatives are similar with GIX PLA₂ of the marine snail Conus magus. Members of GXIV and GIX PLA₂s show modest overall sequence similarity (21–35%) but considerable motif conservation within the putative Ca²⁺-binding, catalytic sites and cysteine residue positions which are essential for enzyme function. GXIV PLA₂s of bacteria and fungi typically contain four cysteine residues composing two intramolecular disulphide bonds. GIX PLA₂ homologues were identified in cnidarians and molluscs and in a single tunicate but appear to be absent from other metazoan genomes. The mature GIX PLA₂ deduced peptides contain up to ten cysteine residues capable of forming five putative disulphide bonds. Three disulphide bonds were identified in GIX PLA₂s, two of which correspond to those localized in GXIV PLA₂s. Phylogenetic analysis demonstrates that metazoan GIX PLA₂s cluster separate from the bacterial and fungal GXIV PLA₂s and both pfam09056 members form a group separate from the prokaryote and eukaryote GXIIA PLA₂ pfam06951. Duplicate PLA₂ pfam09056 genes were identified in the genomes of sea anemone N. vectensis and oyster Crassostrea gigas suggest that members of this family evolved via species-specific duplication events. These observations indicate that the newly identified metazoan pfam09056 members may be classified as GIX PLA₂s and support the idea of the common evolutionary origin of GXIV and GIX PLA₂ pfam09056 members, which emerged early in bacteria and were maintained in the genomes of fungi and selected extant metazoan taxa.     

Mutation in the substrate-binding site of aminopeptidase B confers new enzymatic properties

Aminopeptidase B (Ap-B) catalyzes the cleavage of arginine and lysine residues at the N-terminus of various peptide substrates. In vivo, it participates notably in the miniglucagon and cholecystokinin 8 processing, but the complete range of physiological functions of Ap-B remains to be discovered. Ap-B is a member of the M1 family of Zn²⁺-metallopeptidases that are characterized by two highly conserved motives, GXMEN (potential substrate binding site) and HEXXHX¹⁸E (Zn²⁺-binding site). In this study, mutagenesis and molecular modelling were used to investigate the enzymatic mechanism of Ap-B. Nineteen rat Ap-B mutants of the G₂₉₈XM₃₀₀E₃₀₁N₃₀₂ motif and one mutant of the HEIS₃₂₈HX¹⁸E motif were expressed in Escherichia coli. All mutations except G₂₉₈P, G₂₉₈S, and S₃₂₈A abolished the aminopeptidase activity. The S₃₂₈A mutant mimics the sequence of bovine Ap-B Zn²⁺-binding site, which differs from those of other mammalian Ap-B. This mutant conserved a canonical Ap-B activity. G₂₉₈S and G₂₉₈P mutants exhibit new enzymatic properties such as changes in their profile of inhibition and their sensitivity to Cl⁻ anions. Moreover, the G₂₉₈P mutant exhibits new substrate specificity. A structural analysis using circular dichroism, fluorescence spectroscopy, molecular modelling and dynamics was performed to investigate the role that residue G₂₉₈ plays in the catalytic mechanism of Ap-B. Our results show that G₂₉₈ is essential to Ap-B activity and participates to the substrate specificity of the enzyme.     

In silico and in vitro characterization of phospholipase A2 isoforms from soybean (Glycine max)

At the present, no secreted phospholipase A₂ (sPLA₂) from soybean (Glycine max) was investigated in detail. In this work we identified five sequences of putative secreted sPLA₂ from soybean after a BLAST search in G. max database. Sequence analysis showed a conserved PA2c domain bearing the Ca²⁺ binding loop and the active site motif. All the five mature proteins contain 12 cysteine residues, which are commonly conserved in plant sPLA₂s. We propose a phylogenetic tree based on sequence alignment of reported plant sPLA₂s including the novel enzymes from G. max. According to PLA₂ superfamily, two of G. max sPLA₂s are grouped as XIA and the rest of sequences as XIB, on the basis of differences found in their molecular weights and deviating sequences especially in the N- and C-terminal regions of the isoenzymes. Furthermore, we report the cloning, expression and purification of one of the putative isoenzyme denoted as GmsPLA₂-XIA-1. We demonstrate that this mature sPLA₂ of 114 residues had PLA₂ activity on Triton:phospholipid mixed micelles and determine the kinetic parameters for this system. We generate a model based on the known crystal structure of sPLA₂ from rice (isoform II), giving first insights into the three-dimensional structure of folded GmsPLA₂-XIA-1. Besides describing the spatial arrangement of highly conserved pair HIS-49/ASP-50 and the Ca⁺² loop domains, we propose the putative amino acids involved in the interfacial recognition surface. Additionally, molecular dynamics simulations indicate that calcium ion, besides its key function in the catalytic cycle, plays an important role in the overall stability of GmsPLA₂-XIA-1 structure.     

Efficient catalytic turnover of cytochrome P450(cam) is supported by a T252N mutation

A Thr (or Ser) residue on the I-helix is a highly conserved structural feature of cytochrome P450 enzymes. It is believed to be indispensable as a proton delivery shuttle in the oxygen activation process. Previous work showed that P450_cin (CYP176A1), which contains an Asn instead of the conserved Thr, is fully functional in the catalytic oxidation of cineole [D.B. Hawkes, G.W. Adams, A.L. Burlingame, P.R. Ortiz de Montellano, J.J. De Voss, J. Biol. Chem. 277 (2002) 27725-27732]. To determine whether the substitution of Asn for Thr is specific or general, the conserved Thr252 in P450_cam (CYP101) was mutated to generate the T252N, T252N/V253T, and T252A mutants. Steady-state kinetic analysis of the oxidation of camphor by these mutants indicated that the T252N and T252N/V253T mutants have comparable turnover numbers but higher K_m values relative to the wild-type enzyme. Spectroscopic binding assays indicate that the higher K_m values reflect a decrease in the camphor binding affinity. Non-productive H₂O₂ generation was negligible with the T252N and T252N/V253T mutants, but, as previously observed, was dominant in the T252A mutant. Our results, and a structure model based on the crystal structures of the ferrous dioxygen complexes of P450_cam and its T252A mutant, suggest that Asn252 can stabilize the ferric hydroperoxy intermediate, preventing premature release of H₂O₂ and enabling addition of the second proton to the distal oxygen to generate the catalytic ferryl species.     

Localization and function of cytosolic phospholipase A2α at the Golgi

Cytosolic phospholipase A₂α (cPLA₂α, Group IVA phospholipase A₂) is a central mediator of arachidonate release from cellular phospholipids for the biosynthesis of eicosanoids. cPLA₂α translocates to intracellular membranes including the Golgi in response to a rise in intracellular calcium level. The enzyme’s calcium-dependent phospholipid-binding C2 domain provides the targeting specificity for cPLA₂α translocation to the Golgi. However, other features of cPLA₂α regulation are incompletely understood such as the role of phosphorylation of serine residues in the catalytic domain and the function of basic residues in the cPLA₂α C2 and catalytic domains that are proposed to interact with anionic phospholipids in the membrane to which cPLA₂α is targeted. Increasing evidence strongly suggests that cPLA₂α plays a role in regulating Golgi structure, tubule formation and intra-Golgi transport. For example, recent data suggests that cPLA₂α regulates the transport of tight junction and adherens junction proteins through the Golgi to cell–cell contacts in confluent endothelial cells. However, there are now examples where data based on knockdown using siRNA or pharmacological inhibition of enzymatic activity of cPLA₂α affects fundamental cellular processes yet these phenotypes are not observed in cells from cPLA₂α deficient mice. These results suggest that in some cases there may be compensation for the lack of cPLA₂α. Thus, there is continued need for studies employing highly specific cPLA₂α antagonists in addition to genetic deletion of cPLA₂α in mice.     

A serine point mutation in the adenosine A2AR C-terminal tail reduces receptor heteromerization and allosteric modulation of the dopamine D2R

Evidence exists that the adenosine receptor A_2AR and the dopamine receptor D₂R form constitutive heteromers in living cells. Mass spectrometry and pull-down data showed that an arginine-rich domain of the D₂R third intracellular loop binds via electrostatic interactions to a specific motif of the A_2AR C-terminal tail. It has been indicated that the phosphorylated serine 374 might represent an important residue in this motif. In the present study, it was found that a point mutation of serine 374 to alanine reduced the A_2AR ability to interact with D₂R. Also, this point mutation abolished the A_2AR-mediated inhibition of both the D₂R high affinity agonist binding and signaling. These results point to a key role of serine 374 in the A_2AR-D₂R interface. All together these results indicate that by targeting A_2AR serine 374 it will be possible to allosterically modulate A_2AR-D₂R function, thus representing a new approach for therapeutically modulate D₂R function.     

Emerging roles of secreted phospholipase A2 enzymes: Lessons from transgenic and knockout mice

Among the emerging phospholipase A₂ (PLA₂) superfamily, the secreted PLA₂ (sPLA₂) family consists of low-molecular-mass, Ca²⁺-requiring extracellular enzymes with a His-Asp catalytic dyad. To date, more than 10 sPLA₂ enzymes have been identified in mammals. Individual sPLA₂s exhibit unique tissue and cellular localizations and enzymatic properties, suggesting their distinct pathophysiological roles. Despite numerous enzymatic and cell biological studies on this enzyme family in the past two decades, their precise in vivo functions still remain largely obscure. Recent studies using transgenic and knockout mice for several sPLA₂ enzymes, in combination with lipidomics approaches, have opened new insights into their distinct contributions to various biological events such as food digestion, host defense, inflammation, asthma and atherosclerosis. In this article, we overview the latest understanding of the pathophysiological functions of individual sPLA₂ isoforms fueled by studies employing transgenic and knockout mice for several sPLA₂s.     

Structure–activity relationship studies on 1-heteroaryl-3-phenoxypropan-2-ones acting as inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase: replacement of the activated ketone group by other serine traps

Cytosolic phospholipase A₂α (cPLA₂α) and fatty acid amide hydrolase (FAAH) are serine hydrolases. cPLA₂α is involved in the generation of pro-inflammatory lipid mediators, FAAH terminates the anti-inflammatory effects of endocannabinoids. Therefore, inhibitors of these enzymes may represent new drug candidates for the treatment of inflammation. We have reported that certain 1-heteroarylpropan-2-ones are potent inhibitors of cPLA₂α and FAAH. The serine reactive ketone group of these compounds, which is crucial for enzyme inhibition, is readily metabolized resulting in inactive alcohol derivatives. In order to obtain metabolically more stable inhibitors, we replaced this moiety by α-ketoheterocyle, cyanamide and nitrile serine traps. Investigations on activity and metabolic stability of these substances revealed that in all cases an increased metabolic stability was accompanied by a loss of inhibitory potency against cPLA₂α and FAAH, respectively.     

Effects of light and hydrogen peroxide on gene expression of newly identified antioxidant enzymes in the harmful algal bloom species Chattonella marina

ABSTRACT

Antioxidant enzymes are essential proteins that maintain cell proliferation potential by protecting against oxidative stress. They are present in many organisms including harmful algal bloom (HAB) species. We previously identified the antioxidant enzyme 2-Cys peroxiredoxin (PRX) in the raphidophyte Chattonella marina. This enzyme specifically decomposes a hydrogen peroxide (H₂O₂). PRX is the only antioxidant enzyme so far identified in C. marina. This study used mRNA-seq, using Trinity assemble and blastx for annotation, to identify a further five antioxidant enzymes from C. marina: Cu Zn superoxide dismutase (Cu/Zn-SOD), glutathione peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX) and thioredoxin (TRX). In the gene expression analysis of six enzymes (Cu/Zn-SOD, GPX, CAT, APX, TRX and PRX) using light-acclimated (100 μmol photons m^?2 s^?1) C. marina cells, only PRX gene expression levels were significantly increased by strong light irradiation (1000 μmol photons m^?2 s^?1). H₂O₂ concentration and scavenging activity were also increased and significantly positively correlated with PRX gene expression levels. In dark-acclimated cells, expression levels of all antioxidant enzymes except APX were significantly increased by light irradiation (100 μmol photons m^?2 s^?1). Expression decreased the following day, with the exception of PRX expression. With the exception of CAT, gene expression of antioxidant enzymes was not significantly induced by artificial H₂O₂ treatment, although average gene expression levels were slightly increased in some enzymes. Thus, we suggest that light is the main trigger of gene expression, but the resultant oxidative stress is also a possible factor affecting the gene expression of antioxidant enzymes in C. marina.     

Enzymatic and structural characterization of a basic phospholipase A2 from the sea anemone Condylactis gigantea

This work aimed at the isolation and structural/functional characterization of a phospholipase A₂ (CgPLA₂) from the extract of the anemone Condylactis gigantea. CgPLA₂ was isolated with a high purity level through three chromatographic steps, showing pI ˜ 8.6 and molecular weights of 14,500 and 29,000 for the monomer and dimer, respectively. CgPLA₂ showed a high catalytic activity upon fluorescent phospholipids inducing no direct hemolytic activity. This enzyme, which is Ca²⁺-dependent, showed a lower stability against temperature and pH variations when compared with snake venom enzymes. The enzymatic activity was significantly reduced or completely abolished after chemical modification of CgPLA₂ with BPB. Its cDNA was then obtained, with 357 base pairs which codified for a mature protein of 119 amino acid residues. A comparative analysis of the primary structure of CgPLA₂ revealed 84%, 61%, 43% and 42% similarity to the PLA₂s from Adamsia carciniopados, Nematostella vectensis, Vipera russelli russelli and Bothrops jararacussu, respectively.     

Role of glycine residues highly conserved in the S2-S3 linkers of domains I and II of voltage-gated calcium channel α1 subunits

The pore-forming component of voltage-gated calcium channels, α₁ subunit, contains four structurally conserved domains (I-IV), each of which contains six transmembrane segments (S1-S6). We have shown previously that a Gly residue in the S2-S3 linker of domain III is completely conserved from yeasts to humans and important for channel activity. The Gly residues in the S2-S3 linkers of domains I and II, which correspond positionally to the Gly in the S2-S3 linker of domain III, are also highly conserved. Here, we investigated the role of the Gly residues in the S2-S3 linkers of domains I and II of Ca_v1.2. Each of the Gly residues was replaced with Glu or Gln to produce mutant Ca_v1.2s; G182E, G182Q, G579E, G579Q, and the resulting mutants were transfected into BHK6 cells. Whole-cell patch-clamp recordings showed that current-voltage relationships of the four mutants were the same as those of wild-type Ca_v1.2. However, G182E and G182Q showed significantly smaller current densities because of mislocalization of the mutant proteins, suggesting that Gly¹⁸² in domain I is involved in the membrane trafficking or surface expression of α₁ subunit. On the other hand, G579E showed a slower voltage-dependent current inactivation (VDI) compared to Ca_v1.2, although G579Q showed a normal VDI, implying that Gly⁵⁷⁹ in domain II is involved in the regulation of VDI and that the incorporation of a negative charge alters the VDI kinetics. Our findings indicate that the two conserved Gly residues are important for α₁ subunit to become functional.     

Degumming of vegetable oils by a novel phospholipase B from Pseudomonas fluorescens BIT-18

Pseudomonas fluorescens BIT-18 was isolated from soil near a vegetable oil factory and shown to produce a B-type phospholipase. The enzyme was partially purified by ammonium sulfate precipitation. Gas chromatography demonstrated that the enzyme preparation hydrolyzed both the 1- and 2-ester bonds of phosphatidylcholine. When degumming of soybean, rapeseed, and peanut oil was performed with this enzyme preparation, oils with phosphorous contents lower than 5 mg/kg were obtained after 5 h of enzyme treatment at 40 °C. The enzyme preparation did not show lipase activity, thus free fatty acids were only generated from the phospholipids. Therefore, this novel phospholipase B is potentially useful for the refining of high-quality oils with attractive yields.     

Disturbed brain phospholipid and docosahexaenoic acid metabolism in calcium-independent phospholipase A2-VIA (iPLA2β)-knockout mice

Calcium-independent phospholipase A₂ group VIA (iPLA₂β) releases docosahexaenoic acid (DHA) from phospholipids in vitro. Mutations in the iPLA₂β gene, PLA2G6, are associated with dystonia-parkinsonism and infantile neuroaxonal dystrophy. To understand the role of iPLA₂β in brain, we applied our in vivo kinetic method using radiolabeled DHA in 4 to 5-month-old wild type (iPLA₂β^+/+) and knockout (iPLA₂β^−/−) mice, and measured brain DHA kinetics, lipid concentrations, and expression of PLA₂, cyclooxygenase (COX), and lipoxygenase (LOX) enzymes. Compared to iPLA₂β^+/+ mice, iPLA₂β^−/− mice showed decreased rates of incorporation of unesterified DHA from plasma into brain phospholipids, reduced concentrations of several fatty acids (including DHA) esterified in ethanolamine- and serine-glycerophospholipids, and increased lysophospholipid fatty acid concentrations. DHA turnover in brain phospholipids did not differ between genotypes. In iPLA₂β^−/− mice, brain levels of iPLA₂β mRNA, protein, and activity were decreased, as was the iPLA₂γ (Group VIB PLA₂) mRNA level, while levels of secretory sPLA₂-V mRNA, protein, and activity and cytosolic cPLA₂-IVA mRNA were increased. Levels of COX-1 protein were decreased in brain, while COX-2 protein and mRNA were increased. Levels of 5-, 12-, and 15-LOX proteins did not differ significantly between genotypes. Thus, a genetic iPLA₂β deficiency in mice is associated with reduced DHA metabolism, profound changes in lipid-metabolizing enzyme expression (demonstrating lack of redundancy) and of phospholipid fatty acid content of brain (particularly of DHA), which may be relevant to neurologic abnormalities in humans with PLA2G6 mutations.     

Structural and functional roles of highly conserved serines in human lipoprotein lipase. Evidence that serine 132 is essential for enzyme catalysis

The structure of human lipoprotein lipase was recently deduced from its cDNA sequence. It contains 8 serine residues (residues 45, 132, 143, 172, 193, 244, 251, and 363) that are absolutely conserved in both lipoprotein lipase and hepatic lipase across all species studied. The high homology between lipoprotein lipase, hepatic lipase, and pancreatic lipase suggests that the catalytic functions of these enzymes share a common mechanism and that one of the 8 conserved serines in human lipoprotein lipase must play a catalytic role as does serine 152 in the case of pancreatic lipase (Winkler, F. K., D'Arcy, A., and Hunziker, W. Nature 343, 771-774). We expressed wild-type and site-specific mutants of human lipoprotein lipase in COS cells in vitro. We produced two to four substitution mutants involving each of the 8 serines and assayed a total of 22 mutants for both enzyme activity and the amount of immunoreactive enzyme mass produced. Immunoreactive lipase was detected in all cases. With the exception of Ser132, for each of the 8 serine mutants we studied, at least one of several mutants at each position showed detectable enzyme activity. All three substitution mutants at Ser132, Ser----Thr, Ser----Ala, and Ser----Asp, were totally inactive. Ser132 occurs in the consensus sequence Gly-Xaa-Ser-Xaa-Gly present in all serine proteinases and in human pancreatic lipase. The x-ray crystallography structure of human pancreatic lipase suggests that the analogous serine residue in human pancreatic lipase, Ser152, is the nucleophilic residue essential for catalysis. Our biochemical data strongly support the conclusion that Ser132 in human lipoprotein lipase is the crucial residue required for enzyme catalysis. The observed specific activities of the variants involving the other seven highly conserved serines in human lipoprotein lipase are consistent with the interpretation that this enzyme has a three-dimensional structure very similar to that of human pancreatic lipase.     

Thermotolerance and Related Antioxidant Enzyme Activities Induced by Heat Acclimation and Salicylic Acid in Grape (Vitis vinifera L.) Leaves

Thermotolerance and related antioxidant enzyme activities induced by both heat acclimation and exogenous salicylic acid (SA) application were studied in grapevine (Vitis vinifera L. cv. Jingxiu). Heat acclimation and exogenous SA application induced comparable changes in thermotolerance, ascorbic acid (AsA), glutathione (GSH), and hydrogen peroxide (H₂O₂) concentrations, and in activities of the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), glutathione reductase (GR), ascorbic peroxidase (APX) and catalase (CAT) in grape leaves. Within 1 h at 38 °C, free SA concentration in leaves rose from 3.1 μg g⁻¹ FW to 19.1 μg g⁻¹ FW, then sharply declined. SA application and heat acclimation induced thermotolerance were related to changes of antioxidant enzyme activities and antioxidant concentration, indicating a role for endogenous SA in heat acclimation in grape leaves.     

Plant phospholipases A<Subscript>2</Subscript>: perspectives on biotechnological applications

The recent progress in knowledge on biochemical properties and functions of phospholipases A₂ in plants paved the way for approving the suitability of these enzymes for commercial use now. The secreted phospholipases A₂, representing one type of phospholipases A₂ occurring in plants, show distinct differences in substrate specificities with respect to headgroup and acyl chains of the glycerophospholipids in comparison to their counterparts from animal sources. The other type of phospholipases A₂ in plants, the patatin-related phospholipases A₂, is characterized by broad substrate specificity. Accordingly, the unique properties of the plant enzymes open new horizons to engineered biocatalysts with improved performance, e.g., for vegetable oil refinement by degumming and for targeted modification of phospholipids. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.