Defining the proximal interaction networks of Arf GTPases reveals a mechanism for the regulation of PLD1 and PI4KB.

The Arf GTPase family is involved in a wide range of cellular regulation including membrane trafficking and organelle-structure assembly. Here, we have generated a proximity interaction network for the Arf family using the miniTurboID approach combined with TMT-based quantitative mass spectrometry. Our interactome confirmed known interactions and identified many novel interactors that provide leads for defining Arf pathway cell biological functions. We explored the unexpected finding that phospholipase D1 (PLD1) preferentially interacts with two closely related but poorly studied Arf family GTPases, ARL11 and ARL14, showing that PLD1 is activated by ARL11/14 and may recruit these GTPases to membrane vesicles, and that PLD1 and ARL11 collaborate to promote macrophage phagocytosis. Moreover, ARL5A and ARL5B were found to interact with and recruit phosphatidylinositol 4-kinase beta (PI4KB) at trans-Golgi, thus promoting PI4KB's function in PI4P synthesis and protein secretion.

Zucchini consensus motifs determine the mechanism of pre-piRNA production.

PIWI-interacting RNAs (piRNAs) of between approximately 24 and 31 nucleotides in length guide PIWI proteins to silence transposons in animal gonads, thereby ensuring fertility1. In the biogenesis of piRNAs, PIWI proteins are first loaded with 5'-monophosphorylated RNA fragments called pre-pre-piRNAs, which then undergo endonucleolytic cleavage to produce pre-piRNAs1,2. Subsequently, the 3'-ends of pre-piRNAs are trimmed by the exonuclease Trimmer (PNLDC1 in mouse)3-6 and 2'-O-methylated by the methyltransferase Hen1 (HENMT1 in mouse)7-9, generating mature piRNAs. It is assumed that the endonuclease Zucchini (MitoPLD in mouse) is a major enzyme catalysing the cleavage of pre-pre-piRNAs into pre-piRNAs10-13. However, direct evidence for this model is lacking, and how pre-piRNAs are generated remains unclear. Here, to analyse pre-piRNA production, we established a Trimmer-knockout silkworm cell line and derived a cell-free system that faithfully recapitulates Zucchini-mediated cleavage of PIWI-loaded pre-pre-piRNAs. We found that pre-piRNAs are generated by parallel Zucchini-dependent and -independent mechanisms. Cleavage by Zucchini occurs at previously unrecognized consensus motifs on pre-pre-piRNAs, requires the RNA helicase Armitage, and is accompanied by 2'-O-methylation of pre-piRNAs. By contrast, slicing of pre-pre-piRNAs with weak Zucchini motifs is achieved by downstream complementary piRNAs, producing pre-piRNAs without 2'-O-methylation. Regardless of the endonucleolytic mechanism, pre-piRNAs are matured by Trimmer and Hen1. Our findings highlight multiplexed processing of piRNA precursors that supports robust and flexible piRNA biogenesis.

Structural analysis of PLD3 reveals insights into the mechanism of lysosomal 5' exonuclease-mediated nucleic acid degradation.

The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3.

A Brief Overview of the Toxic Sphingomyelinase Ds of Brown Recluse Spider Venom and Other Organisms and Simple Methods To Detect Production of Its Signature Cyclic Ceramide Phosphate.

A special category of phospholipase D (PLD) in the venom of the brown recluse spider (Loxosceles reclusa) and several other sicariid spiders accounts for the dermonecrosis and many of the other clinical symptoms of envenomation. Related proteins are produced by other organisms, including fungi and bacteria. These PLDs are often referred to as sphingomyelinase Ds (SMase Ds) because they cleave sphingomyelin (SM) to choline and "ceramide phosphate." The lipid product has actually been found to be a novel sphingolipid: ceramide 1,3-cyclic phosphate (Cer1,3P). Since there are no effective treatments for the injury induced by the bites of these spiders, SMase D/PLDs are attractive targets for therapeutic intervention, and some of their features will be described in this minireview. In addition, two simple methods are described for detecting the characteristic SMase D activity using a fluorescent SM analog, (N-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-SM (C12-NBD-SM), that is cleaved to C12-NBD-Cer1,3P, which is easily separated from other potential metabolites by thin-layer chromatography and visualized under UV light. Besides confirming that C12-NBD-Cer1,3P is the only product detected upon incubation of C12-NBD-SM with brown recluse spider venom, the method was also able to detect for the first time very low levels of activity in venom from another spider, Kukulcania hibernalis The simplicity of the methods makes it relatively easy to determine this signature activity of SMase D/PLD. SIGNIFICANCE STATEMENT: The sphingomyelinase D/phospholipase D that are present in the venom of the brown recluse spider and other sources cause considerable human injury, but detection of the novel sphingolipid product, ceramide 1,3-cyclic phosphate, is not easy by previously published methods. This minireview describes simple methods for detection of this activity that will be useful for studies of its occurrence in spider venoms and other biological samples, perhaps including lesions from suspected spider bites and infections.

Phospholipid-Anchored Ligand Conjugation on Extracellular Vesicles for Enhanced Cancer Targeting.

Extracellular vesicles (EVs) are recognized as potential candidates for next-generation drug delivery systems. However, the inherent cancer-targeting efficiency is unsatisfactory, necessitating surface modification to attach cell-binding ligands. By utilizing phospholipase D from Streptomyces in combination with maleimide-containing primary alcohol, the authors successfully anchored ligands onto milk-derived EVs (mEVs), overcoming the issues of ligand leakage or functional alteration seen in traditional methods. Quantitative nano-flow cytometry demonstrated that over 90% of mEVs are effectively modified with hundreds to thousands of ligands. The resulting mEV formulations exhibited remarkable long-term stability in conjugation proportion, ligand number, size distribution, and particle concentration, even after months of storage. It is further shown that conjugating transferrin onto mEVs significantly enhanced cellular uptake and induced pronounced cytotoxic effects when loaded with paclitaxel. Overall, this study presents a highly efficient, stable, cost-effective, and scalable ligand conjugation approach, offering a promising strategy for targeted drug delivery of EVs.

Imaging and Editing the Phospholipidome.

Membranes are multifunctional supramolecular assemblies that encapsulate our cells and the organelles within them. Glycerophospholipids are the most abundant component of membranes. They make up the majority of the lipid bilayer and play both structural and functional roles. Each organelle has a different phospholipid composition critical for its function that results from dynamic interplay and regulation of numerous lipid-metabolizing enzymes and lipid transporters. Because lipid structures and localizations are not directly genetically encoded, chemistry has much to offer to the world of lipid biology in the form of precision tools for visualizing lipid localization and abundance, manipulating lipid composition, and in general decoding the functions of lipids in cells.In this Account, we provide an overview of our recent efforts in this space focused on two overarching and complementary goals: imaging and editing the phospholipidome. On the imaging front, we have harnessed the power of bioorthogonal chemistry to develop fluorescent reporters of specific lipid pathways. Substantial efforts have centered on phospholipase D (PLD) signaling, which generates the humble lipid phosphatidic acid (PA) that acts variably as a biosynthetic intermediate and signaling agent. Though PLD is a hydrolase that generates PA from abundant phosphatidylcholine (PC) lipids, we have exploited its transphosphatidylation activity with exogenous clickable alcohols followed by bioorthogonal tagging to generate fluorescent lipid reporters of PLD signaling in a set of methods termed IMPACT.IMPACT and its variants have facilitated many biological discoveries. Using the rapid and fluorogenic tetrazine ligation, it has revealed the spatiotemporal dynamics of disease-relevant G protein-coupled receptor signaling and interorganelle lipid transport. IMPACT using diazirine photo-cross-linkers has enabled identification of lipid-protein interactions relevant to alcohol-related diseases. Varying the alcohol reporter can allow for organelle-selective labeling, and varying the bioorthogonal detection reagent can afford super-resolution lipid imaging via expansion microscopy. Combination of IMPACT with genome-wide CRISPR screening has revealed genes that regulate physiological PLD signaling.PLD enzymes themselves can also act as tools for precision editing of the phospholipid content of membranes. An optogenetic PLD for conditional blue-light-stimulated synthesis of PA on defined organelle compartments led to the discovery of the role of organelle-specific pools of PA in regulating oncogenic Hippo signaling. Directed enzyme evolution of PLD, enabled by IMPACT, has yielded highly active superPLDs with broad substrate tolerance and an ability to edit membrane phospholipid content and synthesize designer phospholipids in vitro. Finally, azobenzene-containing PA analogues represent an alternative, all-chemical strategy for light-mediated control of PA signaling.Collectively, the strategies described here summarize our progress to date in tackling the challenge of assigning precise functions to defined pools of phospholipids in cells. They also point to new challenges and directions for future study, including extension of imaging and membrane editing tools to other classes of lipids. We envision that continued application of bioorthogonal chemistry, optogenetics, and directed evolution will yield new tools and discoveries to interrogate the phospholipidome and reveal new mechanisms regulating phospholipid homeostasis and roles for phospholipids in cell signaling.

Improvement of solubility of phospholipase D from Streptomyces antibioticus in recombinant Escherichia coli and its application for the enzymatic synthesis of a non-natural plasmalogen.

Plasmalogens are a subclass of glycerophospholipids that have a vinyl-ether bond at the sn-1 position and are thought to have several physiological functions. The creation of non-natural plasmalogens with functional groups is desired for the establishment of the prevention of diseases caused by the depletion of plasmalogens. Phospholipase D (PLD) has both hydrolysis and transphosphatidylation activities. In particular, PLD from Streptomyces antibioticus has been investigated extensively due to its high transphosphatidylation activity. However, it has been difficult to stably express recombinant PLD in Escherichia coli and to express it as a soluble protein. In this study, we used the E. coli strain, SoluBL21™, and achieved stable PLD expression from the T7 promoter and increased soluble fraction in the cell. We also improved the purification method of PLD using His-tag at the C terminus. We obtained PLD with ∼730 mU mg-1 protein of specific activity, and the yield was ∼420 mU l-1 culture, corresponding to 76 mU per gram of wet cells. Finally, we synthesized a non-natural plasmalogen with 1,4-cyclohexanediol bound to the phosphate group at the sn-3 position by transphosphatidylation of the purified PLD. This method will contribute to the expansion of the chemical structure library of non-natural plasmalogens.

Characterization of a phospholipase D from Streptomyces cinnamoneum SK43.003 suitable for phosphatidylserine synthesis.

A phospholipase D high producing strain with transphosphatidylation activity that is suitable for phosphatidylserine synthesis was screened by our laboratory and named as Streptomyces cinnamoneum SK43.003. The enzyme structural and biochemical properties were investigated using the molecular biology method. A 1521-bp fragment of the phospholipase D gene from Streptomyces cinnamoneum SK43.003 was amplified by PCR and encoded for 506 amino acids. The primary structure contained two conserved HKD and GG/S motifs. The pld gene was cloned and expressed in Escherichia coli. The purified enzyme exhibited the highest activity at a pH value of 6.0 andtemperature of 60°C. The enzyme was stable within a pH range of 4-7 for 24 h or at temperatures below 50°C. In addition, Triton X-100, Fe2+ , and Al3+ were beneficial to the enzyme activity, whereas Zn2+ and Cu2+ dramatically inhibited its activity. In a two-phase system, the enzyme could convert phosphatidylcholine to phosphatidylserine with a 92% transformation rate.

Structures of an engineered phospholipase D with specificity for secondary alcohol transphosphatidylation: insights into plasticity of substrate binding and activation.

Phospholipase D (PLD) is an enzyme useful for the enzymatic modification of phospholipids. In the presence of primary alcohols, the enzyme catalyses transphosphatidylation of the head group of phospholipid substrates to synthesise a modified phospholipid product. However, the enzyme is specific for primary alcohols and thus the limitation of the molecular size of the acceptor compounds has restricted the type of phospholipid species that can be synthesised. An engineered variant of PLD from Streptomyces antibioticus termed TNYR SaPLD was developed capable of synthesising 1-phosphatidylinositol with positional specificity of up to 98%. To gain a better understanding of the substrate binding features of the TNYR SaPLD, crystal structures have been determined for the free enzyme and its complexes with phosphate, phosphatidic acid and 1-inositol phosphate. Comparisons of these structures with the wild-type SaPLD show a larger binding site able to accommodate a bulkier secondary alcohol substrate as well as changes to the position of a flexible surface loop proposed to be involved in substrate recognition. The complex of the active TNYR SaPLD with 1-inositol phosphate reveals a covalent intermediate adduct with the ligand bound to H442 rather than to H168, the proposed nucleophile in the wild-type enzyme. This structural feature suggests that the enzyme exhibits plasticity of the catalytic mechanism different from what has been reported to date for PLDs. These structural studies provide insights into the underlying mechanism that governs the recognition of myo-inositol by TNYR SaPLD, and an important foundation for further studies of the catalytic mechanism.

Symmetrically substituted dichlorophenes inhibit N-acyl-phosphatidylethanolamine phospholipase D.

N-Acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) (EC 3.1.4.4) catalyzes the final step in the biosynthesis of N-acyl-ethanolamides. Reduced NAPE-PLD expression and activity may contribute to obesity and inflammation, but a lack of effective NAPE-PLD inhibitors has been a major obstacle to elucidating the role of NAPE-PLD and N-acyl-ethanolamide biosynthesis in these processes. The endogenous bile acid lithocholic acid (LCA) inhibits NAPE-PLD activity (with an IC50 of 68 µm), but LCA is also a highly potent ligand for TGR5 (EC50 0.52 µm). Recently, the first selective small-molecule inhibitor of NAPE-PLD, ARN19874, has been reported (having an IC50 of 34 µm). To identify more potent inhibitors of NAPE-PLD, here we used a quenched fluorescent NAPE analog, PED-A1, as a substrate for recombinant mouse Nape-pld to screen a panel of bile acids and a library of experimental compounds (the Spectrum Collection). Muricholic acids and several other bile acids inhibited Nape-pld with potency similar to that of LCA. We identified 14 potent Nape-pld inhibitors in the Spectrum Collection, with the two most potent (IC50 = ∼2 µm) being symmetrically substituted dichlorophenes, i.e. hexachlorophene and bithionol. Structure-activity relationship assays using additional substituted dichlorophenes identified key moieties needed for Nape-pld inhibition. Both hexachlorophene and bithionol exhibited significant selectivity for Nape-pld compared with nontarget lipase activities such as Streptomyces chromofuscus PLD or serum lipase. Both also effectively inhibited NAPE-PLD activity in cultured HEK293 cells. We conclude that symmetrically substituted dichlorophenes potently inhibit NAPE-PLD in cultured cells and have significant selectivity for NAPE-PLD versus other tissue-associated lipases.

Phosphatidylethanol in whole blood of rhesus monkeys correlates with ethanol consumption.

BACKGROUND: Phosphatidylethanol (PEth) homologs are ethanol metabolites used to identify and monitor alcohol drinking in humans. In this study, we measured levels of the 2 most abundant homologs, PEth 16:0/18:1 and PEth 16:0/18:2, in whole blood samples from rhesus macaque monkeys that drank ethanol daily ad libitum to assess the relationship between PEth levels and recent ethanol exposure in this animal model. METHODS: Blood samples were obtained from The Monkey Alcohol Tissue Research Resource. The monkeys were first induced to consume 4% (w/v) ethanol in water from a panel attached to their home cage. Then, monkeys were allowed to drink ethanol and water ad libitum 22 h daily for 12 months and the daily amount of ethanol each monkey consumed was measured. Whole, uncoagulated blood was collected from each animal at the end of the entire experimental procedure. PEth 16:0/18:1 and PEth 16:0/18:2 levels were analyzed by HPLC with tandem mass spectrometry, and the ethanol consumed during the preceding 14 days was measured. Combined PEth was the sum of the concentrations of both homologs. RESULTS: Our results show that (1) PEth accumulates in the blood of rhesus monkeys after ethanol consumption; (2) PEth homolog levels were correlated with the daily average ethanol intake during the 14-day period immediately preceding blood collection; (3) the application of established human PEth 16:0/18:1 cutoff concentrations indicative of light social or no ethanol consumption (<20 ng/ml), moderate ethanol consumption (≥ 20 and < 200 ng/ml) and heavy ethanol consumption (≥ 200 ng/ml) predicted significantly different ethanol intake in these animals. PEth homologs were not detected in ethanol-naïve controls. CONCLUSIONS: This study confirms that PEth is a sensitive biomarker for ethanol consumption in rhesus macaque monkeys. This nonhuman primate model may prove useful in evaluating sources of variability previously shown to exist between ethanol consumption and PEth homolog levels among humans.

Crystal Structure of a Phospholipase D from the Plant-Associated Bacteria Serratia plymuthica Strain AS9 Reveals a Unique Arrangement of Catalytic Pocket.

Phospholipases D (PLDs) play important roles in different organisms and in vitro phospholipid modifications, which attract strong interests for investigation. However, the lack of PLD structural information has seriously hampered both the understanding of their structure-function relationships and the structure-based bioengineering of this enzyme. Herein, we presented the crystal structure of a PLD from the plant-associated bacteria Serratia plymuthica strain AS9 (SpPLD) at a resolution of 1.79 Å. Two classical HxKxxxxD (HKD) motifs were found in SpPLD and have shown high structural consistence with several PLDs in the same family. While comparing the structure of SpPLD with the previous resolved PLDs from the same family, several unique conformations on the C-terminus of the HKD motif were demonstrated to participate in the arrangement of the catalytic pocket of SpPLD. In SpPLD, an extented loop conformation between ß9 and α9 (aa228-246) was found. Moreover, electrostatic surface potential showed that this loop region in SpPLD was positively charged while the corresponding loops in the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) were neutral. The shortened loop between α10 and α11 (aa272-275) made the SpPLD unable to form the gate-like structure which existed specically in the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) and functioned to stabilize the substrates. In contrast, the shortened loop conformation at this corresponding segment was more alike to several nucleases (Nuc, Zuc, mZuc, NucT) within the same family. Moreover, the loop composition between ß11 and ß12 was also different from the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9), which formed the entrance of the catalytic pocket and were closely related to substrate recognition. So far, SpPLD was the only structurally characterized PLD enzyme from Serratia. The structural information derived here not only helps for the understanding of the biological function of this enzyme in plant protection, but also helps for the understanding of the rational design of the mutant, with potential application in phospholipid modification.

Acyl Chain Specificity of Marine Streptomyces klenkii PhosPholipase D and Its Application in Enzymatic Preparation of Phosphatidylserine.

Mining of phospholipase D (PLD) with altered acyl group recognition except its head group specificity is also useful in terms of specific acyl size phospholipid production and as diagnostic reagents for quantifying specific phospholipid species. Microbial PLDs from Actinomycetes, especially Streptomyces, best fit this process requirements. In the present studies, a new PLD from marine Streptomyces klenkii (SkPLD) was purified and biochemically characterized. The optimal reaction temperature and pH of SkPLD were determined to be 60 °C and 8.0, respectively. Kinetic analysis showed that SkPLD had the relatively high catalytic efficiency toward phosphatidylcholines (PCs) with medium acyl chain length, especially 12:0/12:0-PC (67.13 S-1 mM-1), but lower catalytic efficiency toward PCs with long acyl chain (>16 fatty acids). Molecular docking results indicated that the different catalytic efficiency was related to the increased steric hindrance of long acyl-chains in the substrate-binding pockets and differences in hydrogen-bond interactions between the acyl chains and substrate-binding pockets. The enzyme displayed suitable transphosphatidylation activity and the reaction process showed 26.18% yield with L-serine and soybean PC as substrates. Present study not only enriched the PLD enzyme library but also provide guidance for the further mining of PLDs with special phospholipids recognition properties.

Chemoenzymatic Labeling of Extracellular Vesicles for Visualizing Their Cellular Internalization in Real Time.

Extracellular vesicles (EVs) are intercellular communicators that are heavily implicated in diverse pathological processes. However, it is poorly understood how EVs interact with recipient cells due to the lack of appropriate tracking techniques. Here, we report a robust chemoenzymatic labeling technique for visualizing the internalization process of EVs into target cells in real time. This method uses phospholipase D (PLD) to catalyze the in situ exchange of choline by alkyne in the native EV phosphatidylcholine. Subsequent alkyne-azide click chemistry allows conjugation of Cy5 dyes for visualizing EVs internalization by confocal fluorescence microscopy. The fluorescent labeling of EVs was accomplished in an efficient and biocompatible way, without affecting both the morphology and biological activity of EVs. We applied this chemoenzymatic labeling strategy to monitor the cellular uptake of cancer cell-derived EVs in real time and to further reveal multiple internalization mechanisms. This robust, biocompatible labeling strategy provides an essential tool for EV-related studies ranging from chemical biology to drug delivery.

Assay of Phospholipase D Activity by an Amperometric Choline Oxidase Biosensor.

A novel electrochemical method to assay phospholipase D (PLD) activity is proposed based on the employment of a choline biosensor realized by immobilizing choline oxidase through co-crosslinking on an overoxidized polypyrrole film previously deposited on a platinum electrode. To perform the assay, an aliquot of a PLD standard solution is typically added to borate buffer containing phosphatidylcholine at a certain concentration and the oxidation current of hydrogen peroxide is then measured at the rotating modified electrode by applying a detection potential of + 0.7 V vs. SCE. Various experimental parameters influencing the assay were studied and optimized. The employment of 0.75% (v/v) Triton X-100, 0.2 mM calcium chloride, 5 mM phosphatidylcholine, and borate buffer at pH 8.0, ionic strength (I) 0.05 M allowed to achieve considerable current responses. In order to assure a controlled mass transport and, at the same time, high sensitivity, an electrode rotation rate of 200 rpm was selected. The proposed method showed a sensitivity of 24 (nA/s)(IU/mL)-1, a wide linear range up to 0.33 IU/mL, fast response time and appreciable long-term stability. The limit of detection, evaluated from the linear calibration curve, was 0.005 IU/mL (S/N = 3). Finally, due to the presence of overoxidized polypyrrole film characterized by notable rejection properties towards electroactive compounds, a practical application to real sample analysis can be envisaged.

Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors.

Membranes allow the compartmentalization of biochemical processes and are therefore fundamental to life. The conservation of the cellular membrane, combined with its accessibility to secreted proteins, has made it a common target of factors mediating antagonistic interactions between diverse organisms. Here we report the discovery of a diverse superfamily of bacterial phospholipase enzymes. Within this superfamily, we defined enzymes with phospholipase A1 and A2 activity, which are common in host-cell-targeting bacterial toxins and the venoms of certain insects and reptiles. However, we find that the fundamental role of the superfamily is to mediate antagonistic bacterial interactions as effectors of the type VI secretion system (T6SS) translocation apparatus; accordingly, we name these proteins type VI lipase effectors. Our analyses indicate that PldA of Pseudomonas aeruginosa, a eukaryotic-like phospholipase D, is a member of the type VI lipase effector superfamily and the founding substrate of the haemolysin co-regulated protein secretion island II T6SS (H2-T6SS). Although previous studies have specifically implicated PldA and the H2-T6SS in pathogenesis, we uncovered a specific role for the effector and its secretory machinery in intra- and interspecies bacterial interactions. Furthermore, we find that this effector achieves its antibacterial activity by degrading phosphatidylethanolamine, the major component of bacterial membranes. The surprising finding that virulence-associated phospholipases can serve as specific antibacterial effectors suggests that interbacterial interactions are a relevant factor driving the continuing evolution of pathogenesis.

Phospholipase D engineering for improving the biocatalytic synthesis of phosphatidylserine.

Phosphatidylserine is widely used in food, health, chemical and pharmaceutical industries. The phospholipase D-mediated green synthesis of phosphatidylserine has attracted substantial attention in recent years. In this study, the phospholipase D was heterologously expressed in Bacillus subtilis, Pichia pastoris, and Corynebacterium glutamicum, respectively. The highest activity of phospholipase D was observed in C. glutamicum, which was 0.25 U/mL higher than these in B. subtilis (0.14 U/mL) and P. pastoris (0.22 U/mL). System engineering of three potential factors, including (1) signal peptides, (2) ribosome binding site, and (3) promoters, was attempted to improve the expression level of phospholipase D in C. glutamicum. The maximum phospholipase D activity reached 1.9 U/mL, which was 7.6-fold higher than that of the initial level. The enzyme displayed favorable transphosphatidylation activity and it could efficiently catalyze the substrates L-serine and soybean lecithin for synthesis of phosphatidylserine after optimizing the conversion reactions in detail. Under the optimum conditions (trichloromethane/enzyme solution 4:2, 8 mg/mL soybean lecithin, 40 mg/mL L-serine, and 15 mM CaCl2, with shaking under 40 °C for 10 h), the reaction process showed 48.6% of conversion rate and 1.94 g/L of accumulated phosphatidylserine concentration. The results highlight the use of heterologous expression, system engineering, and process optimization strategies to adapt a promising phospholipase D for efficient phosphatidylserine production in synthetic application.

The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis.

PIWI-family proteins and their associated small RNAs (piRNAs) act in an evolutionarily conserved innate immune mechanism to provide essential protection for germ-cell genomes against the activity of mobile genetic elements. piRNA populations comprise a molecular definition of transposons, which permits them to distinguish transposons from host genes and selectively silence them. piRNAs can be generated in two distinct ways, forming either primary or secondary piRNAs. Primary piRNAs come from discrete genomic loci, termed piRNA clusters, and seem to be derived from long, single-stranded precursors. The biogenesis of primary piRNAs involves at least two nucleolytic steps. An unknown enzyme cleaves piRNA cluster transcripts to generate monophosphorylated piRNA 5' ends. piRNA 3' ends are probably formed by exonucleolytic trimming, after a piRNA precursor is loaded into its PIWI partner. Secondary piRNAs arise during the adaptive 'ping-pong' cycle, with their 5' termini being formed by the activity of PIWIs themselves. A number of proteins have been implicated genetically in primary piRNA biogenesis. One of these, Drosophila melanogaster Zucchini, is a member of the phospholipase-D family of phosphodiesterases, which includes both phospholipases and nucleases. Here we produced a dimeric, soluble fragment of the mouse Zucchini homologue (mZuc; also known as PLD6) and show that it possesses single-strand-specific nuclease activity. A crystal structure of mZuc at 1.75 Å resolution indicates greater architectural similarity to phospholipase-D family nucleases than to phospholipases. Together, our data suggest that the Zucchini proteins act in primary piRNA biogenesis as nucleases, perhaps generating the 5' ends of primary piRNAs.

Effect of N- and C-Terminal Amino Acids on the Interfacial Binding Properties of Phospholipase D from Vibrio parahaemolyticus.

The effects of N-terminal (1⁻34 amino acids) and C-terminal (434⁻487 amino acids) amino acid sequences on the interfacial binding properties of Phospholipase D from Vibrio parahaemolyticus (VpPLD) were characterized by using monomolecular film technology. Online tools allowed the prediction of the secondary structure of the target N- and C-terminal VpPLD sequences. Various truncated forms of VpPLD with different N- or C-terminal deletions were designed, based on their secondary structure, and their membrane binding properties were examined. The analysis of the maximum insertion pressure (MIP) and synergy factor "a" indicated that the loop structure (1⁻25 amino acids) in the N-terminal segment of VpPLD had a positive effect on the binding of VpPLD to phospholipid monolayers, especially to 1,2-dimyristoyl-sn-glycero-3-phosphoserine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The deletion affecting the N-terminus loop structure caused a significant decrease of the MIP and synergy factor a of the protein for these phospholipid monolayers. Conversely, the deletion of the helix structure (26⁻34 amino acids) basically had no influence on the binding of VpPLD to phospholipid monolayers. The deletion of the C-terminal amino acids 434⁻487 did not significantly change the binding selectivity of VpPLD for the various phospholipid monolayer tested here. However, a significant increase of the MIP value for all the phospholipid monolayers strongly indicated that the three-strand segment (434⁻469 amino acids) had a great negative effect on the interfacial binding to these phospholipid monolayers. The deletion of this peptide caused a significantly greater insertion of the protein into the phospholipid monolayers examined. The present study provides detailed information on the effect of the N- and C-terminal segments of VpPLD on the interfacial binding properties of the enzyme and improves our understanding of the interactions between this enzyme and cell membranes.

Active site mapping of Loxosceles phospholipases D: Biochemical and biological features.

Brown spider phospholipases D from Loxosceles venoms are among the most widely studied toxins since they induce dermonecrosis, triggering inflammatory responses, increase vascular permeability, cause hemolysis, and renal failure. The catalytic (H12 and H47) and metal-ion binding (E32 and D34) residues in Loxosceles intermedia phospholipase D (LiRecDT1) were mutated to understand their roles in the observed activities. All mutants were identified using whole venom serum antibodies and a specific antibody to wild-type LiRecDT1, they were also analyzed by circular dichroism (CD) and differential scanning calorimetry (DSC). The phospholipase D activities of H12A, H47A, H12A-H47A, E32, D34 and E32A-D34A, such as vascular permeability, dermonecrosis, and hemolytic effects were inhibited. The mutant Y228A was equally detrimental to biochemical and biological effects of phospholipase D, suggesting an essential role of this residue in substrate recognition and binding. On the other hand, the mutant C53A-C201A reduced the enzyme's ability to hydrolyze phospholipids and promote dermonecrosis, hemolytic, and vascular effects. These results provide the basis understanding the importance of specific residues in the observed activities and contribute to the design of synthetic and specific inhibitors for Brown spider venom phospholipases D.