Distinct Substrate Specificities Research Articles

Study of The Divergent Acyl Chain Specificities of The Class I Ceramide Synthases in Plants

Ceramide synthases catalyze the condensation of long-chain bases and fatty acyl-CoA to form the ceramide backbones of glycosphingolipids.The Class I and II ceramide synthases of plants have distinct substrate specificities that contribute to the divergent fatty acid chain-lengths and functions of plant sphingolipids. The Arabidopsis LOH2-encoded Class I ceramide synthase has been shown to have strong specificity for palmitoyl (16:0)-CoA that gives rise to ceramides with C16 fatty acids that are prevalent in dicot glucosylceramides. By contrast, glucosylceramides of many monocots and selected dicots are enriched in C18 and C20-ceramides. We hypothesized that these differences are due to functional and structural differences in LOH2 ceramide synthases. Consistent with this, we found that transgenic expression of a tomato LOH2 restored C16 fatty acid in glucosylceramides in an Arabidopsis loh2 mutant, whereas expression of a rice LOH2 resulted in C18- and C20-fatty acid-enriched glucosylceramides. To establish the structural basis for these different outcomes, we demonstrated that swapping a divergent 16 amino acid domain from the rice LOH2 to tomato LOH2 yielded C18- and C20-fatty acid-glucosylceramides when expressed in the loh2 mutant. By comparison of primary structures of LOH2 enzymes from diverse dicots and monocots, we identified a single amino acid that when mutated in the tomato LOH2 resulted in rice-type C18- and C20 fatty acid-glucosylceramides.We rationalize the altered LOH2 function in the context of the predicted LOH2 ceramide synthase secondary structure. Overall, our findings highlight the biochemical and genetic basis for divergent ceramide structures in dicots and monocots.

Role of the F‐box protein exchange factor in the reproductive growth of Arabidopsis

Cullin-Associated NEDD8 Dissociated Protein 1 (CAND1) is one of the key factors regulating the Skp1-Cul1-F-box protein (SCF) ubiquitin ligases. SCFs are enzyme complexes catalyzing the ubiquitination reaction, which tags cellular proteins with a chain of ubiquitin and designates them for subsequent degradation. In this way, SCFs can specify the half-lives of important regulatory proteins, and therefore, they play essential roles in numerous biological processes, such as hormone signaling, cell cycle regulation, immune response, and organ development. All SCFs have a core Cul1 scaffold protein, and it assembles with a member from the F-box protein family to form different SCFs with distinct substrate specificity. Akin to an exchange factor, CAND1 functions by increasing the rate at which an F-box protein dissociates from Cul1 and thus facilitating the recycling of the limited Cul1 scaffold of SCF complexes. The discovery of the CAND1 working mechanism has led to the adaptive exchange hypothesis, in which CAND1-mediated exchange enables cells to tolerate varying levels of F-box proteins and ensures the assembly of the right type of SCF at the right time for timely substrate ubiquitination. To test this hypothesis, an F-box protein was over-expressed in cultured human cells with or without the CAND1 gene, and cells lacking CAND1 became inviable upon the F-box protein over-expression while the normal cells were unaffected. This result provides the first evidence supporting the hypothesis, and it suggests that a CAND1-defective multicellular organism may fail to maintain cell types that contain a large pool of F-box proteins. Thus, we further investigated the role of CAND1 in the development of a multicellular organism. While the CAND1 knockout mice are reported to be homozygous lethal, homozygous cand1 knockout mutant Arabidopsis are viable, exhibiting impaired growth and development at various stages. Particularly, cand1 mutant Arabidopsis are almost sterile, and our studies with cell biological, physiological and bioinformatic analyses have shown that the diminished fertility was due to the death of pollen, which contains the largest pool of F-box protein transcripts among all analyzed tissues. These findings are consistent with results from studies using cultured human cells, and they provide natural evidence supporting the adaptive exchange hypothesis.

Specific reduction of a 1‐Cys Prx from Pseudomonas aeruginosa by ascorbate

Pseudomonas aeruginosa is a ubiquitous gammaproteobacteria that is the major cause of hospitalar infections of pneumonia and is the major pathogen associated with lung infections in cystic fibrosis patients. Among several defense systems against pathogens, host cells release oxidants, such as: superoxide (O2˙-), hydrogen peroxide (H2O2) and peroxynitrite during phagocytosis. To overcome this oxidative insult, P. aeruginosa possesses a myriad of strategies, including Cys-based peroxidases called peroxiredoxins. One of the peroxiredoxins from P. aeruginosa is LsfA that belongs to the Prx6 subgroup, containing only one cysteine and displays the so-called 1-Cys Prx mechanism. In addition, LsfA was previously related with P. aeruginosa virulence. Therefore, the aim of the present work is to characterize LsfA in biochemical and structural grounds. Here, we describe two crystallographic structures of LsfA in distinct oxidative states: reduced (Cys-S-) and sulfonic acid (Cys-SO3H) at 2.6 and 2.0 Ǻ resolution, respectively. The native dimeric state was confirmed by crystallography and SAXS (Small angle X ray scattering) in the reduced (Cys-S-), oxidized (Cys-SOH) and hyperoxidized (Cys-SO2/3H) states. The LsfA active site topology is slightly different than the human Prx6 homologue, suggesting that these enzymes might present distinct substrate specificities. Furthermore, other differences were observed, such as the orientation of His37 that changes in a redox dependent manner in human Prx6, but not in LsfA. We also investigated the substrates specificities of LsfA. Previously, we observed that LsfA decomposes distinct peroxides at extremely high rates (k = 106 - 107 M-1.s-1). However, a reductant is required to turnover LsfA, but the identity of this compound is still unknown to LsfA. Therefore, we investigated if thioredoxin or glutathione/glutaredoxin systems from P. aeruginosa could act as LsfA reductants. We employed recombinant enzymes and coupled assays and we observed that the thioredoxin or glutathione/glutaredoxin systems failed to support the peroxidase activity of LsfA. Previously, we observed that ascorbate reduces the sulfenic acid form of LsfA with a second order constant rate of 103 M-1.s-1, using a competition assay based on DCPIP (2,6–dichlorophenolindophenol). Therefore, we investigated the effects of ascorbate on P. aeruginosa growth, employing a bacterial strain with the LsfA gene deleted. Wild type and ΔlsfA strains did not grew on ascorbate as a sole carbon source. Furthermore, no ascorbate toxicity up to 300 mM concentration was observed, when growing both strains in a minimum media with glucose as carbon source. Studies are underway to understand how P. aeruginosa cells uptake and export ascorbate. Furthermore, the dependencies of P. aeruginosa cells on LsfA and ascorbate to decompose exogenously added peroxides will be investigated.

New Avenues for Phosphodiesterase Inhibitors in Asthma.

IntroductionPhosphodiesterases (PDEs) are isoenzymes ubiquitously expressed in the lungs where they catalyse cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (GMP), which are fundamental second messengers in asthma, thereby regulating the intracellular concentrations of these cyclic nucleotides, their signaling pathways and, consequently, myriad biological responses. The superfamily of PDEs is composed of 11 families with a distinct substrate specificity, molecular structure and subcellular localization. Experimental studies indicate a possible role in asthma mainly for PDE3, PDE4, PDE5 and PDE7. Consequently, drugs that inhibit PDEs may offer novel therapeutic options for the treatment of this disease.Areas CoveredIn this article, we describe the progress made in recent years regarding the possibility of using PDE inhibitors in the treatment of asthma.Expert OpinionMany data indicate the potential benefits of PDE inhibitors as an add-on treatment especially in severe asthma due to their bronchodilator and/or anti-inflammatory activity, but no compound has yet reached the market as asthma treatment mainly because of their limited tolerability. Therefore, there is a growing interest in developing new PDE inhibitors with an improved safety profile. In particular, the research is focused on the development of drugs capable of interacting simultaneously with different PDEs, or to be administered by inhalation. CHF 6001 and RPL554 are the only molecules that currently are under clinical development but there are several new agents with interesting pharmacological profiles. It will be stimulating to assess the impact of such agents on individual treatable traits in specially designed studies.

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms.

Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homolog of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson's mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, the interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we observe that maximal stimulation of the TBK1 or PINK1 pathway does not elevate Rab7A phosphorylation. Altogether, these findings reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.

Phospho-Form Specific Substrates of Protein Kinase B (AKT1).

Protein kinase B (AKT1) is hyper-activated in diverse human tumors. AKT1 is activated by phosphorylation at two key regulatory sites, Thr308 and Ser473. Active AKT1 phosphorylates many, perhaps hundreds, of downstream cellular targets in the cytosol and nucleus. AKT1 is well-known for phosphorylating proteins that regulate cell survival and apoptosis, however, the full catalog of AKT1 substrates remains unknown. Using peptide arrays, we recently discovered that each phosphorylated form of AKT1 (pAKT1S473, pAKT1T308, and ppAKT1S473,T308) has a distinct substrate specificity, and these data were used to predict potential new AKT1 substrates. To test the high-confidence predictions, we synthesized target peptides representing putative AKT1 substrates. Peptides substrates were synthesized by solid phase synthesis and their purity was confirmed by mass spectrometry. Most of the predicted peptides showed phosphate accepting activity similar to or greater than that observed with a peptide derived from a well-established AKT1 substrate, glycogen synthase kinase 3β (GSK-3β). Among the novel substrates, AKT1 was most active with peptides representing PIP3-binding protein Rab11 family-interacting protein 2 and cysteinyl leukotriene receptor 1, indicating their potential role in AKT1-dependent cellular signaling. The ppAKT1S473,T308 enzyme was highly selective for peptides containing a patch of basic residues at −5, −4, −3 and aromatic residues (Phe/Tyr) at +1 positions from the phosphorylation site. The pAKT1S473 variant preferred more acidic peptides, Ser or Pro at +4, and was agnostic to the residue at −5. The data further support our hypothesis that Ser473 phosphorylation plays a key role in modulating AKT1 substrate selectivity.

Transcriptome and Secretome Analysis of Intra-Mammalian Life-Stages of Calicophoron daubneyi Reveals Adaptation to a Unique Host Environment

AbstractParamphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock, which has seen a rapid rise in prevalence throughout Western Europe in recent years. After ingestion of metacercariae (parasite cysts) by the mammalian host, newly excysted juveniles (NEJs) emerge and invade the duodenal submucosa, which causes significant pathology in heavy infections. The immature flukes then migrate upward, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients, and avoid the host immune response. Here, transcriptome analysis of four intramammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic diseases, respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that while a family of cathepsins B with varying S2 subsite residues (indicating distinct substrate specificities) is differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is upregulated in adult worms, although they are under-represented in the secretome. The most abundant proteins in adult fluke secretions were helminth defense molecules that likely establish an immune environment permissive to fluke survival and/or neutralize pathogen-associated molecular patterns such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognize antigens from other helminths commonly found as coinfections with rumen fluke.

Crystal structure of the catalytic domain of botulinum neurotoxin subtype A3

Botulinum neurotoxins (BoNTs) are among the most widely used therapeutic proteins; however, only two subtypes within the seven serotypes, BoNT/A1 and BoNT/B1, are currently used for medical and cosmetic applications. Distinct catalytic properties, substrate specificities, and duration of enzymatic activities potentially make other subtypes very attractive candidates to outperform conventional BoNTs in particular therapeutic applications. For example, BoNT/A3 has a significantly shorter duration of action than other BoNT/A subtypes. Notably, BoNT/A3 is the subtype with the least conserved catalytic domain among BoNT/A subtypes. This suggests that the sequence differences, many of which concern the α-exosite, contribute to the observed functional differences in toxin persistence by affecting the binding of the substrate SNAP-25 and/or the stability of the catalytic domain fold. To identify the molecular determinants accounting for the differences in the persistence observed for BoNT/A subtypes, we determined the crystal structure of the catalytic domain of BoNT/A3 (LC/A3). The structure of LC/A3 was found to be very similar to that of LC/A1, suggesting that the overall mode of SNAP-25 binding is common between these two proteins. However, circular dichroism (CD) thermal unfolding experiments demonstrated that LC/A3 is significantly less stable than LC/A1, implying that this might contribute to the reduced toxin persistence of BoNT/A3. These findings could be of interest in developing next-generation therapeutic toxins.

Translating the Concept of Bispecific Antibodies to Engineering Heterodimeric Phosphotriesterases with Broad Organophosphate Substrate Recognition.

We have adopted the concept of bispecific antibodies, which can simultaneously block or cross-link two different biomolecular targets, to create bispecific enzymes by exploiting the homodimeric quaternary structure of bacterial phosphotriesterases (PTEs). The PTEs from Brevundimonas diminuta and Agrobacterium radiobacter, whose engineered variants can efficiently hydrolyze organophosphorus (OP) nerve agents and pesticides, respectively, have attracted considerable interest for the treatment of the corresponding intoxications. OP nerve agents and pesticides still pose a severe toxicological threat in military conflicts, including acts of terrorism, as well as in agriculture, leading to >100000 deaths per year. In principle, engineered conventional homodimeric PTEs may provoke hydrolytic inactivation of individual OPs in vivo, and their application as catalytic bioscavengers via administration into the bloodstream has been proposed. However, their narrow substrate specificity would necessitate therapeutic application of a set or mixture of different enzymes, which complicates biopharmaceutical development. We succeeded in combining subunits from both enzymes and to stabilize their heterodimerization by rationally designing electrostatic steering mutations, thus breaking the natural C2 symmetry. The resulting bispecific enzyme from two PTEs with different bacterial origin exhibits an ultrabroad OP substrate profile and allows the efficient detoxification of both nerve agents and pesticides. Our approach of combining two active sites with distinct substrate specificities within one artificial dimeric biocatalyst-retaining the size and general properties of the original enzyme without utilizing protein mixtures or much larger fusion proteins-not only should facilitate biological drug development but also may be applicable to oligomeric enzymes with other catalytic activities.

Chemistry, biosynthesis and biological activity of terpenoids and meroterpenoids in bacteria and fungi isolated from different marine habitats.

The marine environment with its vast biological diversity encompasses many organisms that produce bioactive natural products. Marine microorganisms are rich sources of compounds from many structural classes with a multitude of biological activities. The biosynthesis of microbial natural products depends on a variety of biotic and abiotic factors in the marine environment, including temperature, nutrients, salinity and interaction with other microorganisms. Terpenoids, as one of the most important groups of natural products in terrestrial microorganisms are important metabolites for marine microorganisms. Here, we have reviewed the chemistry, biosynthesis and pharmacological activities of terpenoids, extracted from marine microbes, and then survey their potential applications in drug development. We also discussed the different habitats in which marine microorganisms are found including sediments, the flora, such as seaweeds, sea grasses, and mangroves as well as the fauna like sponges and corals. Amongst these habitats, marine sediments are the major source for terpenoids producing microorganisms. The marine bacteria produce mostly meroterpenoids, while the fungi are well known for production of isoprenoids. Interestingly, marine-derived microbial terpenoids have some structural characteristics such as halogenation, which are catalyzed by specific enzymes with distinct substrate specificity. These compounds have anticancer, antibacterial, antifungal, antimalarial and anti-inflammatory properties. The information collected here might provide useful clues for developing new medications.

Correction to: Proteases from the marine bacteria in the genus Pseudoalteromonas: diversity, characteristics, ecological roles, and application potentials

Bacterial strains of the genus Pseudoalteromonas, which includes 48 species, are widely distributed in various ocean environments. Many strains are often abundant and the highest protease producer among marine culturable protease-secreting bacteria, suggesting their important role in marine organic nitrogen degradation and cycling. The extracellular proteases of Pseudoalteromonas are diverse, including serine proteases, metalloproteases, and cystine proteases. Pseudoalteromonas proteases have unique properties, such as cold adaptation, salt tolerance, distinct substrate specificity, and catalytic mechanism. They play important ecological roles in marine organic nitrogen degradation and in the interactions of Pseudoalteromonas with other organisms. Some Pseudoalteromonas proteases have shown promising application potentials in bioactive peptide preparation and meat processing. In this review, advances in our knowledge of Pseudoalteromonas proteases are introduced, with a focus on diversity, characters, ecological significance, and application potentials.

Deletion of the pps-like gene activates the cryptic phaC genes in Haloferax mediterranei.

Haloferax mediterranei, a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) producing haloarchaeon, possesses four PHA synthase encoding genes, phaC, phaC1, phaC2, and phaC3. In the wild-type strain, except phaC, the other three genes are cryptic and not transcribed under PHA-accumulating conditions. The PhaC protein together with PhaE subunit forms the active PHA synthase and catalyzes PHBV polymerization. Previously, it was observed that the deletion of a gene named pps-like significantly enhanced PHBV accumulation probably resulted from the upregulation of pha cluster genes (phaR-phaP-phaE-phaC). The present study demonstrated the influence of pps-like gene deletion on the cryptic phaC genes. As revealed by qRT-PCR, the expression level of the three cryptic genes was upregulated in the ΔEPSΔpps-like geneΔphaC mutant. Sequential knockout of the cryptic phaC genes and fermentation experiments showed that PhaC1 followed by PhaC3 had the ability to synthesize PHBV in ΔEPSΔpps-like geneΔphaC mutant. Both PhaC1 and PhaC3 could complex with PhaE to form functionally active PHA synthase. However, the expression of phaC2 did not lead to PHBV synthesis. Moreover, PhaC, PhaC1, and PhaC3 exhibited distinct substrate specificity as the 3HV content in PHBV copolymers was different. The EMSA result showed that PPS-like protein might be a negative regulator of phaC1 gene by binding to its promoter region. Taken together, PhaC1 had the most pronounced effect on PHBV synthesis in ΔEPSΔpps-like geneΔphaC mutant and deletion of pps-like gene released the negative effect from phaC1 expression and thereby restored PHBV accumulating ability in ΔphaC mutant. KEY POINTS: • Cryptic phaC genes were activated by pps-like gene deletion. • PPS-like protein probably regulated phaC1 expression by binding to its promoter. • Both PhaC1 and PhaC3 formed active PHA synthase with PhaE.

Characterization of two family AA9 LPMOs from Aspergillus tamarii with distinct activities on xyloglucan reveals structural differences linked to cleavage specificity.

Aspergillus tamarii grows abundantly in naturally composting waste fibers of the textile industry and has a great potential in biomass decomposition. Amongst the key (hemi)cellulose-active enzymes in the secretomes of biomass-degrading fungi are the lytic polysaccharide monooxygenases (LPMOs). By catalyzing oxidative cleavage of glycoside bonds, LPMOs promote the activity of other lignocellulose-degrading enzymes. Here, we analyzed the catalytic potential of two of the seven AA9-type LPMOs that were detected in recently published transcriptome data for A. tamarii, namely AtAA9A and AtAA9B. Analysis of products generated from cellulose revealed that AtAA9A is a C4-oxidizing enzyme, whereas AtAA9B yielded a mixture of C1- and C4-oxidized products. AtAA9A was also active on cellopentaose and cellohexaose. Both enzymes also cleaved the β-(1→4)-glucan backbone of tamarind xyloglucan, but with different cleavage patterns. AtAA9A cleaved the xyloglucan backbone only next to unsubstituted glucosyl units, whereas AtAA9B yielded product profiles indicating that it can cleave the xyloglucan backbone irrespective of substitutions. Building on these new results and on the expanding catalog of xyloglucan- and oligosaccharide-active AA9 LPMOs, we discuss possible structural properties that could underlie the observed functional differences. The results corroborate evidence that filamentous fungi have evolved AA9 LPMOs with distinct substrate specificities and regioselectivities, which likely have complementary functions during biomass degradation.

A tale of non-canonical tails: gene regulation by post-transcriptional RNA tailing.

RNA tailing, or the addition of non-templated nucleotides to the 3' end of RNA, is the most frequent and conserved type of RNA modification. The addition of tails and their composition reflect RNA maturation stages and have important roles in determining the fate of the modified RNAs. Apart from canonical poly(A) polymerases, which add poly(A) tails to mRNAs in a transcription-coupled manner, a family of terminal nucleotidyltransferases (TENTs), including terminal uridylyltransferases (TUTs), modify RNAs post-transcriptionally to control RNA stability and activity. The human genome encodes 11 different TENTs with distinct substrate specificity, intracellular localization and tissue distribution. In this Review, we discuss recent advances in our understanding of non-canonical RNA tails, with a focus on the functions of human TENTs, which include uridylation, mixed tailing and post-transcriptional polyadenylation of mRNAs, microRNAs and other types of non-coding RNA.

Biochemical and structural insights into an Ochrobactrum sp. CSL1 ribose-5-phosphate isomerase A and its roles in isomerization of rare sugars

Rare sugars have received increasing attention due to their important applications as sweeteners and building blocks. The substrate specificity and catalytic properties of ribose-5-phosphate isomerase A (RpiA) in isomerization of rare sugars have not been extensively explored. In this study, an RpiA from Ochrobactrum sp. CSL1 was cloned and expressed in Escherichia coli. The biochemical and reaction features were explored and its broad substrate specificity was identified. A higher reaction rate in isomerizing l-rhamnose to l-rhamnulose by OsRpiA, compared with OsRpiB found in the same strain indicated higher efficiency in preparing rare sugars, which was verified by kinetics study. The 2.8 Å resolution structure of OsRpiA was then solved and used in subsequent molecular dynamics experiments, providing a possible explanation for its distinct substrate specificity. The present study highlighted the unique role of microbial RpiA in preparing rare sugars, and its structural information provided a reliable reference for further reaction mechanism research and enzyme engineering work.

Feruloyl Esterases for Biorefineries: Subfamily Classified Specificity for Natural Substrates.

Feruloyl esterases (FAEs) have an important role in the enzymatic conversion of lignocellulosic biomass by decoupling plant cell wall polysaccharides and lignin. Moreover, FAEs release anti-oxidative hydroxycinnamic acids (HCAs) from biomass. As a plethora of FAE candidates were found in fungal genomes, FAE classification related to substrate specificity is an indispensability for selection of most suitable candidates. Hence, linking distinct substrate specificities to a FAE classification, such as the recently classified FAE subfamilies (SF), is a promising approach to improve the application of these enzymes for a variety of industrial applications. In total, 14 FAEs that are classified members of SF1, 5, 6, 7, 9, and 13 were tested in this research. All FAEs were investigated for their activity toward a variety of substrates: synthetic model substrates, plant cell wall-derived substrates, including lignin, and natural substrates. Released HCAs were determined using reverse phase-ultra high performance liquid chromatography coupled to UV detection and mass spectrometry. Based on this study, FAEs of SF5 and SF7 showed the highest release of FA, pCA, and diFAs over the range of substrates, while FAEs of SF6 were comparable but less pronounced for diFAs release. These results suggest that SF5 and SF7 FAEs are promising enzymes for biorefinery applications, like the production of biofuels, where a complete degradation of the plant cell wall is desired. In contrast, SF6 FAEs might be of interest for industrial applications that require a high release of only FA and pCA, which are needed as precursors for the production of biochemicals. In contrast, FAEs of SF1, 9 and 13 showed an overall low release of HCAs from plant cell wall-derived and natural substrates. The obtained results substantiate the previous SF classification as a useful tool to predict the substrate specificity of FAEs, which eases the selection of FAE candidates for industrial applications.

Unique Substrate Specificity of Human Phospholipase A2s

Phospholipase A2 (PLA2) constitutes a superfamily of enzymes that hydrolyze the sn‐2 acyl‐chain of phospholipids which are the major components of cell membranes. PLA2 produces free fatty acids and lysophospholipids as products which serve as lipid mediators and precursors of bioactive lipids. Among them, the release of ω‐6 and ω‐3 fatty acids including arachidonic acid (AA, ω‐6), eicosapentaenoic acid (EPA, ω‐3), and docosahexaenoic acid (DHA, ω‐3) is a pivotal role of PLA2s because the fatty acids are precursors of various eicosanoids and related bioactive oxylipins responsible for inflammation and resolution. Additionally, in contrast to conventional ester‐linked phospholipids, we also investigated PLA2 specificity toward sn‐1 vinyl ether‐linked phospholipids and phosphatidylinositol phosphates (PIPs) which are involved in various signaling processes.Cytosolic PLA2 (cPLA2), calcium‐independent PLA2 (iPLA2), and secreted PLA2 (sPLA2) are the three major PLA2s that are well studied, and a significant number of PLA2‐related disorders have been reported. Therefore, investigating the substrate specificity of PLA2 toward different phospholipids, can be a key to understanding the etiology of PLA2‐related disorders and developing novel therapeutic targets. Recently, we developed a high‐throughput lipidomics‐based assay to measure PLA2 activity and successfully employed it to investigate the general specificity of PLA2s (1). In the present study, we investigated the specificity of the three major PLA2s toward phospholipids containing ω‐6 and ω‐3 fatty acids in the sn‐2 position, sn‐1 vinyl ethers, and PIPs using this lipidomics‐based assay. Also, we determined the optimal binding of the substrate in the active site of each PLA2 by molecular dynamics (MD) simulations to explain the observed specificity.cPLA2 showed high activity toward AA and EPA, and prefers sn‐1 vinyl ether phospholipids over ester phospholipids. iPLA2 showed the highest activity toward EPA, and no preference was observed between a vinyl ether and an ester. sPLA2 showed a preference for DHA and ester phospholipids. In addition, we determined that although additional phosphates on the inositol headgroup attenuated the activity of all three PLA2s, at least cPLA2 and iPLA2 could hydrolyze PI(4,5)P2. MD simulations revealed that the active site of cPLA2 could precisely bind AA and EPA in the sn‐2 position but not DHA. In summary, the present study revealed a unique and distinct substrate specificity for each of the PLA2s and explained the observed preference of cPLA2 for AA and EPA.Support or Funding InformationSupported by NIH grant GM20501‐43

The Four Arabidopsis Choline/Ethanolamine Kinase Isozymes Play Distinct Roles in Metabolism and Development.

Phosphatidylcholine and phosphatidylethanolamine are two major phospholipid classes in eukaryotes. Each biosynthesis pathway starts with the phosphorylation of choline (Cho) or ethanolamine (Etn) catalyzed by either choline or ethanolamine kinase (CEK). Arabidopsis contains four CEK isoforms, but their isozyme-specific roles in metabolism and development are poorly described. Here, we showed that these four CEKs have distinct substrate specificities in vitro. While CEK1 and CEK2 showed substrate preference for Cho over Etn, CEK3 and CEK4 had clear substrate specificity for Cho and Etn, respectively. In vivo, CEK1, CEK2, and CEK3 exhibited kinase activity for Cho but not Etn, although the latter two isoforms showed rather minor contributions to total Cho kinase activity in both shoots and roots. The knockout mutants of CEK2 and CEK3 both affected root growth, and these isoforms had nonoverlapping cell-type-specific expression patterns in the root meristematic zone. In-depth phenotype analysis, as well as chemical and genetic complementation, revealed that CEK3, a Cho-specific kinase, is involved in cell elongation during root development. Phylogenetic analysis of CEK orthologs in Brassicaceae species showed evolutionary divergence between Etn kinases and Cho kinases. Collectively, our results demonstrate the distinct roles of the four CEK isoforms in Cho/Etn metabolism and plant development.

The expression, purification, and substrate analysis of matrix metalloproteinases in Drosophila melanogaster

Matrix metalloproteinases (MMPs) are evolutionarily conserved extracellular matrix proteinases. Genetic analysis of the Drosophila MMPs, Mmp1 and Mmp2, in vivo reveal that they play vital roles in tissue remodeling. Although the catalytic domain (CD) undertakes most MMP functions, few studies have sought to demonstrate the biochemical properties of the CDs of fly MMPs. Here, we identified the overexpression, purification, and refolding of the CDs of Drosophila Mmp1 and Mmp2 for biochemical studies. Zymography assays and substrate degradation analysis showed that both Mmp1-CD and Mmp2-CD were able to digest casein, gelatin, fibronectin, collagen (types I, IV, and V), while Mmp2-CD showed much higher degradation activity compared with Mmp1-CD. Moreover, human collagen III could be degraded by Mmp1-CD but not Mmp2-CD, and rat collagen I and laminin could be degraded by Mmp2-CD but not Mmp1-CD, suggesting that Drosophila Mmp1 and Mmp2 might have overlapping yet distinct substrate specificity. Using synthetic fluorescent substrates, we further demonstrated that the enzymatic activity of Mmp1-CD and Mmp2-CD could be inhibited by human tissue inhibitors of metalloproteinases (TIMPs). These results reveal the context of the cooperative yet distinct roles of Mmp1 and Mmp2 in tissue remodeling.

Function of ceramide synthases on growth, ganoderic acid biosynthesis and sphingolipid homeostasis in Ganoderma lucidum

Ceramide synthases (CERSs) catalyse an N-acyltransferase reaction using long-chain base (LCB) and fatty acyl-coenzyme A (CoA) as substrates to synthesize ceramide (Cer), which is the backbone of all complex sphingolipids. In the present study, three CERSs (LAG1, LAG2 and LAG3) form Ganoderma lucidum were analysed. The silencing of lag1 by RNA interference reduced ganoderic acid biosynthesis and Cer and complex sphingolipids contents, which contain long-chain-fatty-acids (LCFAs, including C16 and C18). In contrast, the silencing of lag2 or lag3 did not result in obvious phenotypic and sphingolipid homeostasis changes, although the lag2/lag3 double-silenced mutants exhibited increased ganoderic acid biosynthesis as well as reduced growth, reduced Cer and complex sphingolipids contents, which contain very-long-chain fatty acids (VLCFAs, including C22, C24 and C26). The results of the present study indicate that the three assayed CERSs have distinct physiological functions and substrate specificities in G. lucidum.