Identification and biochemical characterization of a GDSL-motif carboxylester hydrolase from Carica papaya latex

An esterase (CpEst) showing high specific activities on tributyrin and short chain vinyl esters was obtained from Carica papaya latex after an extraction step with zwitterionic detergent and sonication, followed by gel filtration chromatography. Although the protein could not be purified to complete homogeneity due to its presence in high molecular mass aggregates, a major protein band with an apparent molecular mass of 41 kDa was obtained by SDS-PAGE. This material was digested with trypsin and the amino acid sequences of the tryptic peptides were determined by LC/ESI/MS/MS. These sequences were used to identify a partial cDNA (679 bp) from expressed sequence tags (ESTs) of C. papaya. Based upon EST sequences, a full-length gene was identified in the genome of C. papaya, with an open reading frame of 1029 bp encoding a protein of 343 amino acid residues, with a theoretical molecular mass of 38 kDa. From sequence analysis, CpEst was identified as a GDSL-motif carboxylester hydrolase belonging to the SGNH protein family and four potential N-glycosylation sites were identified. The putative catalytic triad was localised (Ser³⁵-Asp³⁰⁷-His³¹⁰) with the nucleophile serine being part of the GDSL-motif. A 3D-model of CpEst was built from known X-ray structures and sequence alignments and the catalytic triad was found to be exposed at the surface of the molecule, thus confirming the results of CpEst inhibition by tetrahydrolipstatin suggesting a direct accessibility of the inhibitor to the active site.     

Purification, Characterization and Cloning of Phospholipase D from Peanut Seeds

We purified phospholipase D (PLD) enzyme from peanut seeds, and the PLD enzyme eluted as two distinct peak fractions on Mono-Q chromatography, the first of which was characterized. N-terminal sequencing indicated that the N-terminus was blocked. The molecular mass of the purified enzyme was estimated to be 92 kDa by SDS-PAGE. The pH optimum of the enzyme was 5.0, and the K _m value against its substrate phosphatidylcholine (PC), in the presence of 10 mM CaCl₂ and 4 mM deoxycholate, was estimated to be 0.072 mM. The enzyme catalyzed two reactions, i.e., hydrolysis of PC generating phosphatidic acid (PA) and choline, and transphosphatidylation of the PA-moiety in the PC molecule to the acceptor glycerol, generating phosphatidylglycerol. Furthermore, we cloned two types of full-length cDNA, Ahpld1 and Ahpld2, each encoding distinct PLD molecules having 794 and 807 residues, respectively. The partial amino acid sequence of the purified PLD was consistent with the deduced sequence of AhPLD2.     

C-terminal loop of Streptomyces phospholipase D has multiple functional roles

We have recently shown that two flexible loops of Streptomyces phospholipase D (PLD) affect the catalytic reaction of the enzyme by a comparative study of chimeric PLDs. Gly188 and Asp191 of PLD from Streptomyces septatus TH-2 (TH-2PLD) were identified as the key amino acid residues involved in the recognition of phospholipids. In the present study, we further investigated the relationship between a C-terminal loop of TH-2PLD and PLD activities to elucidate the reaction mechanism and the recognition of the substrate. By analyzing chimeras and mutants in terms of hydrolytic and transphosphatidylation activities, Ala426 and Lys438 of TH-2PLD were identified as the residues associated with the activities. We found that Gly188 and Asp191 recognized substrate forms, whereas residues Ala426 and Lys438 enhanced transphosphatidylation and hydrolysis activities regardless of the substrate form. By substituting Ala426 and Lys438 with Phe and His, respectively, the mutant showed not only higher activities but also higher thermostability and tolerance against organic solvents. Furthermore, the mutant also improved the selectivity of the transphosphatidylation activity. The residues Ala426 and Lys438 were located in the C-terminal flexible loop of Streptomyces PLD separate from the highly conserved catalytic HxKxxxxD motifs. We demonstrated that this C-terminal loop, which formed the entrance of the active well, has multiple functional roles in Streptomyces PLD.     

Bioconversion of Phosphatidylserine by Phospholipase D from Streptomyces racemochromogenes in a Microaqueous Water-Immiscible Organic Solvent

Phospholipase D (PLD)-mediated transphosphatidylation of phosphatidylcholine (PC) in a biphasic system was limited by the hydrolysis reaction. A biphasic system can produce a large amount of water. To solve this problem, a microaqueous water-immiscible organic solvent was used for the first time in the bioconversion of phosphatidylserine (PS). The transphosphatidylation among 40 µmol PC, 800 µmol L-serine, and 0.17 U/mL PLD in 2.133 mL of butyl acetate with 6.25% water (V/V) was conducted at a trans-phosphatidylation rate of 88% (mol/mol), and no hydrolytic reaction was observed. Compared to commonly used biphasic systems, this system shows a similar transphosphatidylation rate, whereas the undesirable hydrolysis of phospholipids was completely suppressed.     

Kinetics of myocardial phospholipase D

Myocardial phospholipase D (PLD) is located in different subcellular membranes, including sarcolemma (SL) and sarcoplasmic reticulum (SR). In this study, the kinetics of PLD-dependent hydrolytic and transphosphatidylation activities were examined in SL and SR fractions isolated from rat heart by measuring the formation of phosphatidic acid and phosphatidylethanol, respectively. The results showed that, compared to SR PLD, SL PLD had a higher V_max, i.e. 373 vs. 70 nmol/mg protein/h for the hydrolytic activity and 415 vs. 60 nmol/mg protein/h for the transphosphatidylation activity. In comparison with the SR enzyme, SL PLD had a lower K_m value for the hydrolytic activity (0.46 vs. 0.65 mM), but a higher K_m for the transphosphatidylation activity (225 vs. 179 mM). These distinctive kinetic parameters suggest that SL PLD and SR PLD may be isoforms of the enzyme and/or have different membrane domain. Therefore, SL- and SR-localized PLD activities may be under independent control mechanism(s) and play distinct roles in normal conditions and pathological processes.     

The transphosphatidylation activity of phospholipase D

Transphosphatidylation activity is a characteristic and remarkable property of phospholipase D (PLD) and has been studied in plants and mammalian tissues. This reaction is often used to confirm the properties and/or abnormalities of PLD activity. The mechanism for activating PLD transphosphatidylation seems multiple. Although significant changes of transphosphatidylation activity have been found in some pathological animal models, the biological significance of PLD transphosphatidylation remains largely unknown.     

Substrate preference of stress-activated phospholipase D in Chlamydomonas and its contribution to PA formation

In response to various environmental stress conditions, plants rapidly form the intracellular lipid second messenger phosphatidic acid (PA). It can be generated by two independent signalling pathways via phospholipase D (PLD) and via phospholipase C (PLC) in combination with diacylglycerol kinase (DGK). In the green alga Chlamydomonas, the phospholipid substrates for these pathways are characterized by specific fatty acid compositions. This allowed us to establish: (i) PLD's in vivo substrate preference; and (ii) PLD's contribution to PA formation during stress signalling. Accordingly, G-protein activation (1 micro m mastoparan), hyperosmotic stress (150 mm NaCl) and membrane depolarization (50 mm KCl) were used to stimulate PLD, as monitored by the accumulation in 5 min of its unique transphosphatidylation product phosphatidylbutanol (PBut). In each case, PBut's fatty acid composition specifically matched that of phosphatidylethanolamine (PE), identifying this lipid as PLD's favoured substrate. This conclusion was substantiated by analysing the molecular species by electrospray ionization-mass spectrometry (ESI-MS/MS), which revealed that PE and NaCl-induced PBut share a unique (18 : 1)2-structure. The fatty acid composition of PA was much more complex, reflecting the different contributions from the PLC/DGK and PLD pathways. During KCl-induced stress, the PA rise was largely accounted for by PLD activity. In contrast, PLD's contribution to hyperosmotic stress-induced PA was less, being approximately 63% of the total increase. This was because the PLC/DGK pathway was activated as well, resulting in phosphoinositide-specific fatty acids and molecular species in PA.     

Phospholipase D from Streptoverticillium cinnamoneum: protein engineering and application for phospholipid production

This review is focusing on an industrially important enzyme, phospholipase D (PLD), exhibiting both transphosphatidylation and hydrolytic activities for various phospholipids. The transphosphatidylation activity of PLD is particularly useful for converting phosphatidylcholine (PC) into other phospholipids. During the last decade, the genes coding for PLD have been identified from various species including mammals, plants, yeast, and bacteria. However, detailed basic and applied enzymological studies on PLD have been hampered by the low productivity in these organisms. Efficient production of a recombinant PLD has also been unsuccessful so far. We recently isolated and characterized the PLD gene from Streptoverticillium cinnamoneum, producing a secretory PLD. Furthermore, we constructed an overexpression system for the secretory enzyme in an active and soluble form using Streptomyces lividans as a host for transformation of the PLD gene. The Stv. cinnamoneum PLD was proven to be useful for the continuous and efficient production of phosphatidylethanolamine (PE) from phosphatidylcholine. Thus, the secretory PLD is a promising catalyst for synthesizing new phospholipids possessing various polar head groups that show versatile physiological functions and may be utilized in food and pharmaceutical industries.     

A comparison between continuous and batch processes to produce phosphatidylserine in the efficient and green aqueous-solid system

The efficient and environmentally friendly aqueous-solid systems employed Triton-X-100-modified silica as the “artificial interface” to adsorb phosphatidylcholine (PC) in purely aqueous solutions and silica-adsorbed PC was successfully used for phospholipase D (PLD)-mediated transphosphatidylation. Three kinds of silicas with different sizes were employed to investigate advantages and disadvantages of batch and continuous technologies for PLD-catalyzed transphosphatidylation in aqueous-solid systems. The highest yields of product were obtained in the batch technology, but the continuous production had the simplest operational process and highest space–time yield. After transphosphatidylation, the product adsorbed on carriers were eluted by coconut oil and used to manufacture relevant hard, soft, and micro-capsules. Special attention has been paid to the preparation of microcapsules. Toxic solvents were completely avoided in the whole technological process including production and product packaging. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2777, 2019.     

A facile transphosphatidylation reaction using a culture supernatant of actinomycetes directly as a phospholipase D catalyst with a chelating agent

An attempt was made to use the phospholipase D (PLD)- containing culture supernatants of actinomycetes directly as catalysts for the transphosphatidylation reaction of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) in a biphasic system. Of the five actinomycetes (three Streptomyces sp. and two Streptoverticillium sp.) examined, three (St. mediocidicus, Stv. cinnamoneum and Stv. hachijoense) exhibited good PLD production performance, but the selectivity (ratio of transphosphatidylation to hydrolysis) of the PLDs in the culture supernatant of all three actinomycetes were significantly low. However, the addition of EDTA to the reaction mixture as a chelating agent remarkably improved the selectivity of the PLDs, which approached 100% in all the culture supernatants. Commercially available PLDs were also investigated and classified into two types. The PLDs of one type had high selectivity and no metal was required for the enzyme activity, while those of the other type showed low selectivity and a metal was necessary for the enzyme to be activated. From this finding, it was considered that the culture supernatants used in this study contained several PLDs of both types. When the chelating agent was added to the reaction mixture, the hydrolysis due to PLDs with low selectivity was suppressed by removal of the essential metal, resulting in an increased in the overall selectivity of the PLDs in the culture supernatant. Repeated batch transphosphatidylation reactions were performed 20 times, reusing the PLDs in the aqueous phase by centrifugation; the reaction rate gradually decreased to 60% of that of batch 1 by batch 20. This suggests that the transphosphatidylation reaction using a culture supernatant has potential for industrial application. (c) 1994 John Wiley & Sons, Inc.     

Two highly homologous phospholipase D isoenzymes from Papaver somniferum L. with different transphosphatidylation potential

The genes of two phospholipase D (PLD) isoenzymes, PLD1 and PLD2, from poppy seedlings (2829 and 2828 bp) were completely sequenced. The two genes have 96.9% identity in the encoding region and can be assigned to the alpha-type of plant PLDs. The corresponding amino acid sequences do not contain any signal sequences. One Asn-glycosylation site, six and two phosphorylation sites for protein kinase C and tyrosine kinase, respectively, and two phosphatidylinositol-4,5-bisphosphate binding motifs could be identified. Like in most plant PLDs, two HKD motifs and one C2 domain are present. PLD1 and PLD2 have ten and nine cysteine residues. The two enzymes were expressed in E. coli and purified to homogeneity by Ca2+ ion-mediated hydrophobic interaction chromatography. The Ca2+ ion concentration needed for carrier binding of the two enzymes in chromatography as well as for optimum activity was found to be considerably higher (>100 mM) than with other alpha-type plant PLDs. Although PLD1 and PLD2 differ in eleven amino acids only, they showed remarkable differences in their transphosphatidylation activity. Two amino acid exchanges within and near the first HKD motif contribute to this difference as shown by the A349E/E352Q-variant of PLD2.     

Purification and characterization of a wound-inducible thaumatin-like protein from the latex of Carica papaya

A 22.137 kDa protein constituent of fresh latex was isolated both from the latex of regularly damaged papaya trees and from a commercially available papain preparation. The protein was purified up to apparent homogeneity and was shown to be absent in the latex of papaya trees that had never been previously mechanically injured. This suggests that the protein belongs to pathogenesis-related protein family, as expected for several other protein constituents of papaya latex. The protein was identified as a thaumatin-like protein (class 5 of the pathogenesis-related proteins) on the basis of its partial amino acid sequence. By sequence analysis of the Carica genome, three different forms of thaumatin-like protein were identified, where the latex constituent belongs to a well-known form, allowing the molecular modeling of its spatial structure. The papaya latex thaumatin-like protein was further characterized. The protein appears to be stable in the pH interval from 2 to 10 and resistant to chemical denaturation by guanidium chloride, with a of 15.2 kcal/mol and to proteolysis by the four papaya cysteine proteinases. The physiological role of this protein is discussed.     

Identification of a putative triacylglycerol lipase from papaya latex by functional proteomics

Latex from Caricaceae has been known since 1925 to contain strong lipase activity. However, attempts to purify and identify the enzyme were not successful, mainly because of the lack of solubility of the enzyme. Here, we describe the characterization of lipase activity of the latex of Vasconcellea heilbornii and the identification of a putative homologous lipase from Carica papaya. Triacylglycerol lipase activity was enriched 74-fold from crude latex of Vasconcellea heilbornii to a specific activity (SA) of 57 μmol·min(-1)·mg(-1) on long-chain triacylglycerol (olive oil). The extract was also active on trioctanoin (SA = 655 μmol·min(-1)·mg(-1) ), tributyrin (SA = 1107 μmol·min(-1)·mg(-1) ) and phosphatidylcholine (SA = 923 μmol·min(-1)·mg(-1) ). The optimum pH ranged from 8.0 to 9.0. The protein content of the insoluble fraction of latex was analyzed by electrophoresis followed by mass spectrometry, and 28 different proteins were identified. The protein fraction was incubated with the lipase inhibitor [(14) C]tetrahydrolipstatin, and a 45 kDa protein radiolabeled by the inhibitor was identified as being a putative lipase. A C. papaya cDNA encoding a 55 kDa protein was further cloned, and its deduced sequence had 83.7% similarity with peptides from the 45 kDa protein, with a coverage of 25.6%. The protein encoded by this cDNA had 35% sequence identity and 51% similarity to castor bean acid lipase, suggesting that it is the lipase responsible for the important lipolytic activities detected in papaya latex.     

Cloning analysis of ferritin and the cisplatin-subunit for cancer cell apoptosis in Aplysia juliana hepatopancreas

Ferritin, an iron storage protein, plays a key role in iron metabolism in vivo. Here, we have cloned an inducible ferritin cDNA with 519 bp within the open reading frame fragment from the hepatopancreas of Aplysia juliana (AJ). The subunit sequence of the ferritin was predicted to be a polypeptide of 172 amino acids with a molecular mass of 19.8291kDa and an isoelectric point of 5.01. The cDNA sequence of hepatopancreas ferritin in AJ was constructed into a pET-32a system for expressing its relative protein efficiently in E. coli strain BL21, under isopropyl-β-d-thiogalactoside induction. The recombinant ferritin, which was further purified on a Ni-NTA resin column and digested with enterokinase, was detected as a single subunit of approximately 20 kDa mass using both SDS-PAGE and mass spectrometry. The secondary structure and phosphorylation sites of the deduced amino acids were predicted using both ExPASy proteomic tools and the NetPhos 2.0 server, and the subunit space structure of the recombinant AJ ferritin (rAjFer) was built using a molecular operating environment software system. The result of in-gel digestion and identification using MALDI-TOF MS/MS showed that the recombinant protein was AjFer. ICP-MS results indicated that the rAjFer subunit could directly bind to cisplatin[cis-Diaminedichloroplatinum(CDDP)], giving approximately 17.6 CDDP/ferritin subunits and forming a novel CDDP-subunit. This suggests that a nanometer CDDP core-ferritin was constructed, which could be developed as a new anti-cancer drug. The flow cytometry results indicated that CDDP-rAjFer could induce Hela cell apoptosis. Results of the real-time PCR and Western blotting showed that the expression of AjFer mRNA was up-regulated in AJ under Cd(2+) stress. The recombinant AjFer protein should prove to be useful for further study of the structure and function of ferritin in Aplysia.     

Phospholipid and triacylglycerol profiles modified by PLD suppression in soybean seed

Phospholipase D (PLD) is capable of hydrolyzing membrane phospholipids, producing phosphatidic acid. To alter phospholipid profiles in soybean seed, we attenuated PLD enzyme activity by an RNA interference construct using the partial sequence from a soybean PLDα gene. Two transgenic soybean lines were established by particle inflow gun (PIG) bombardment by co‐bombarding with pSPLDi and pHG1 vectors. The lines were evaluated for the presence and expression of transgenes thoroughly through the T₄ generation. PLD‐suppressed soybean lines were characterized by decreased PLDα enzyme activity and decreased PLDα protein both during seed development and in mature seeds. There was no change in total phospholipid amount; however, the PLD‐attenuated transgenic soybean seed had higher levels of di18 : 2 (dilinoleoyl)‐phosphatidylcholine (PC) and ‐phosphatidylethanolamine (PE) in seeds than the non‐transgenic lines. The increased polyunsaturation was at the expense of PC and PE species containing monounsaturated or saturated fatty acids. In addition to increased unsaturation in the phospholipids, there was a decrease in unsaturation of the triacylglycerol (TAG) fraction of the soybean seeds. Considering recent evidence for the notion that desaturation of fatty acids occurs in the PC fraction and that the PC → DAG (diacylglycerol) → TAG pathway is the major route of TAG biosynthesis in developing soybean seed, the current data suggest that PLDα suppression slows the conversion of PC to TAG. This would be consistent with PLD playing a positive role in that conversion. The data indicate that soybean PLD attenuation is a potentially useful approach to altering properties of edible and industrial soybean lecithin.     

Efficient and green aqueous−solid system for transphosphatidylation to produce phosphatidylhydroxybutyrate: Potential drugs for central nervous system's diseases

The synthesis of nonnatural phospholipid, phosphatidylhydroxybutyrate (PB), was firstly introduced by phospholipase D (PLD)-mediated transphosphatidylation of phosphatidylcholine (PC) with sodium γ-hydroxybutyrate (NaGHB) in the aqueous–solid system. Nanoscale silicon dioxide (NSD) was employed as a carrier to provide an “artificial interphase” between PC and PLD. Special attention has been paid to the effect of the PC coverage on the surface area of hybrids of NSD-PC, the PC loading and the yield of PB. Results indicated that the highest PC loading of 98.3% and the highest PB yield of 97.3% were achieved. In addition, the free PLD in the aqueous–solid system showed the greater stability and pH tolerance than that in the traditional liquid–liquid system. The operational stability of free PLD solution was investigated. The yield of PB remained 70.7% after being used for five batches. The authors provide a new idea for drug design and the potential source of PB for medical experiments. PB is a potential drug and may have the excellent performance in the treatment of central nervous system's diseases. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2726, 2019     

A novel low molecular weight phospholipase D from Streptomyces sp. CS684

With the aim of isolating economically viable enzymes from a microbial source, a novel phospholipase D (PLD) was purified from Streptomyces sp. CS684 (PLD(684)). PLD(684) had molecular weight of 29 kDa, which makes it the second smallest PLD reported so far. The enzyme activity was optimum at pH 6 and 45 degrees C, and enhanced by various detergents. It was stable from pH 7 to 9 and at or below 45 degrees C when assayed after 40 h and 2h, respectively. The K(m) and V(max) values for phosphatidylcholine were 1.16 mM and 1453.74 micromol min(-1)mg(-1), respectively. It catalyzed the transphosphatidylation of glycerol, but not that of l-serine, myo-inositol or ethanolamine. Low molecular weight PLD(684) with transphosphatidylation activity may be utilized in the industrial production of rare and commercially important phospholipids.     

A HPLC-fluorescence detection method for determination of cardiac phospholipase D activity in vitro

A nonradioactive assay for the investigation of phospholipase D (PLD) activity in cardiac membranes has been developed. A fluorescent derivative of phosphatidylcholine [2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3proprionyl)amino) undecyl) sn-glycero-3-phosphocholine] was utilized as substrate in an in vitro PLD-catalyzed transphosphatidylation reaction utilizing ethanol as second substrate. Unreacted phosphatidylcholine and the products of phospholipase activity (PEtOH, phosphatidylethanol; PA, phosphatidic acid; DAG, diacylglycerol) were separated by a binary gradient HPLC system and detected by fluorometry. The detection limit of this assay is approximately 0.6 pmol PEtOH. The reaction proceeded at a linear rate for up to 45 min and increased linearly with increasing amounts of rat cardiac membrane protein in a range of 0.625 microg up to at least 25 microg. In the presence of potassium fluoride, formation of fluorescent PA increased at the expense of DAG generation, demonstrating the presence of PA phosphohydrolase activity in rat cardiac membranes. PEtOH formation was unchanged in the presence of the PA phosphohydrolase inhibitor, indicating that the phosphatidylalcohol is not subject to further metabolism by this enzyme. Activation of protein kinase C by phorbol ester significantly increased PLD activity in cardiac membranes. This assay proved to be sensitive for accurate and rapid assessment of PLD activity in cardiac membranes permitting further characterization of the regulation of PLD signal transduction in the heart.     

Expression, characterization, and crystallization of a member of the novel phospholipase D family of phosphodiesterases.

A family of phospholipase D (PLD) proteins has recently been identified (Koonin, 1996; Ponting & Kerr, 1996) based upon amino acid sequence identity. This family includes human and plant PLDs, proteins encoded by open reading frames in pathogenic viruses and bacteria, as well as an endonuclease. The endonuclease, known as Nuc, is encoded by the IncN plasmid, pKM101, present in Salmonella typhimurium. The recombinant Nuc protein has been expressed and purified from Escherichia coli. The amino-terminal sequencing of the purified protein indicated that the mature protein started from the 23rd residue of the predicted sequence, suggesting that the protein is proteolytically processed during export to the periplasmic space. The recombinant enzyme was able to hydrolyze both double and single-strand DNA and an artificial substrate, bis(4-nitrophenyl) phosphate, which contains a phosphodiester bond. The enzyme activity was not inhibited in the presence of EDTA and was not regulated by divalent cations. The purified protein has been crystallized by hanging drop vapor diffusion methods, and those crystals diffract to 1.9 A resolution.