Profile of the in silico secretome of the palm dieback pathogen, a fungus that puts natural oases at risk

Understanding biotic changes that occur alongside climate change constitute a research priority of global significance. Here, we address a plant pathogen that poses a serious threat to life on natural oases, where climate change is already taking a toll and severely impacting human subsistence. Fusarium oxysporum f. sp. albedinis is a pathogen that causes dieback disease on date palms, a tree that provides several critical ecosystem services in natural oases; and consequently, of major importance in this vulnerable habitat. Here, we assess the current state of global pathogen spread, we annotate the genome of a sequenced pathogen strain isolated from the native range and we analyse its in silico secretome. The palm dieback pathogen secretes a large arsenal of effector candidates including a variety of toxins, a distinguished profile of secreted in xylem proteins (SIX) as well as an expanded protein family with an N-terminal conserved motif [SG]PC[KR]P that could be involved in interactions with host membranes. Using agrobiodiversity as a strategy to decrease pathogen infectivity, while providing short term resilient solutions, seems to be widely overcome by the pathogen. Hence, the urgent need for future mechanistic research on the palm dieback disease and a better understanding of pathogen genetic diversity.

Recent Advanced Metabolic and Genetic Engineering of Phenylpropanoid Biosynthetic Pathways

The MYB transcription factors (TFs) are evolving as critical role in the regulation of the phenylpropanoid and tanshinones biosynthetic pathway. MYB TFs relate to a very important gene family, which are involved in the regulation of primary and secondary metabolisms, terpenoids, bioactive compounds, plant defense against various stresses and cell morphology. R2R3 MYB TFs contained a conserved N-terminal domain, but the domain at C-terminal sorts them different regarding their structures and functions. MYB TFs suppressors generally possess particular repressive motifs, such as pdLNLD/ELxiG/S and TLLLFR, which contribute to their suppression role through a diversity of complex regulatory mechanisms. A novel flower specific “NF/YWSV/MEDF/LW” conserved motif has a great potential to understand the mechanisms of flower development. In the current review, we summarize recent advanced progress of MYB TFs on transcription regulation, posttranscriptional, microRNA, conserved motif and propose directions to future prospective research. We further suggest there should be more focus on the investigation for the role of MYB TFs in microalgae, which has great potential for heterologous protein expression system for future perspectives.

Protein Kinase CK2 Phosphorylates a Conserved Motif in the Notch Effector E(spl)-Mγ

Across metazoans, the effects of Notch signaling are mediated via the Enhancer of Split (E(spl)/HES) basic Helix-Loop-Helix-Orange (bHLH-O) repressors. Although conserved, sequence diversity is, in large part, restricted to the C-terminal domain (CtD), which separates the O-domain from the penultimate WRPW motif that binds the co-repressor Groucho. While the kinases CK2 and MAPK target the CtD and regulate Drosophila E(spl)-M8 and mammalian HES6, the generality of this regulation to other E(spl)/HES repressors has remained unknown. To determine the broader impact of phosphorylation on this large family of repressors, we conducted bioinformatics, evolutionary and biochemical analyses. Our studies identify E(spl)-Mγ as a new target of native CK2 purified from Drosophila embryos, reveal that phosphorylation is specific to CK2 and independent of the regulatory CK2-β subunit, and identify that the site of phosphorylation is juxtaposed to the WRPW motif, a feature unique to and conserved in the Mγ homologues over 50x106 years of Drosophila evolution. Thus, a preponderance of E(spl) homologues in Drosophila are targets for CK2, and the distinct positioning of the CK2 and MAPK sites, raises the prospect that phosphorylation underlies functional diversity of bHLH-O proteins.

Butyryl/Caproyl-CoA:Acetate CoA-Transferase: Cloning, Expression and Characterization of the Key Enzyme Involved in Medium-Chain Fatty Acid Biosynthesis

Coenzyme A transferases (CoATs) are important enzymes involved in carbon chain elongation, contributing to medium-chain fatty acid (MCFA) biosynthesis. For example, butyryl-CoA:acetate CoA transferase (BCoAT) is responsible for the final step of butyrate synthesis from butyryl-CoA. However, little is known about caproyl-CoA:acetate CoA-transferase (CCoAT), which is responsible for the final step of caproate synthesis from caproyl-CoA. In this study, two CoAT genes from Ruminococcaceae bacterium CPB6 and Clostridium tyrobutyricum BEY8 were identified by gene cloning and expression analysis. Enzyme assays and kinetic studies were carried out using butyryl-CoA or caproyl-CoA as the substrate. CPB6-CoAT can catalyze the conversion of both butyryl-CoA to butyrate and caproyl-CoA to caproate, but its catalytic efficiency with caproyl-CoA as the substrate was 3.8 times higher than that with butyryl-CoA. In contrast, BEY8-CoAT had only BCoAT activity, not CCoAT activity. This demonstrated the existence of a specific CCoAT involved in chain elongation via the reverse β-oxidation pathway. Comparative bioinformatics analysis showed the presence of a highly conserved motif (GGQXDFXXGAXX) in CoATs, which is predicted to be the active center. Single point mutations in the conserved motif of CPB6-CoAT (Asp346 and Ala351) led to marked decreases in the activity for butyryl-CoA and caproyl-CoA, indicating that the conserved motif is the active center of CPB6-CoAT and that Asp346 and Ala351 have a significant impact on the enzymatic activity. This work provides insight into the function of CCoAT in caproic acid biosynthesis and improves understanding of the chain elongation pathway for MCFA production.

Bioinformatics Analysis and Identification of Phytoene Synthase Gene in Microalgae

Background: Carotenoids are known as lipophilic secondary metabolites with important biological activities, which are mostly used in the food and pharmaceutical industry. They contribute to the colours of many fruits and flowers. Studies on the biosynthetic pathways of isoprenoids and carotenoids are still scarce, especially in microalgae, and are limited to specific groups Dunaliella spp. In the Chlorophyta taxon of algae, the 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate (DOXP/MEP) is the synthesis pathway of sterols and carotenoids. Objectives: In this study, we used 12 Psy gene sequences in Dunaliella sp., also Scenedesmus acutus, and Diospyros kaki to investigate a genome-wide search. The results are useful for better identification of carotenoids metabolisms and increasing the production rate of beta-carotene in pharmaceutical, food, and industrial processes. Methods: Phytoene synthase (Psy) from Dunaliella spp. was selected as the first regulatory point in the carotenoids pathway that catalysis the formation of geranylgeranyl pyrophosphate in isoprenoid biosynthesis. Structural, evolutionary, and physics-chemical characteristics were investigated using various bioinformatics tools and computer techniques. Moreover, some recently published patents were also regarded. Results: The maximum length of the conserved motif was 5167 bp for Dunaliella. sp. (DQ463306.1), and the smallest length of the conserved motif was 416 bp belong to D. salina (JQ762451.1). The average molecular weight of species was 41820.53 Da. The theoretical pI of species varied from 4.87 to 9.65, indicating vernation in the acidic nature. Two strains of D. bardawil (U91900.1 and EU328287.1) showed just a long-distance relationship with all other Dunaliella strains. Whilst, D. parva displayed the furthest vicinity with all the studied strains. Conclusion: Our study highlighted the Psy regulatory mechanism as a key factor in the carotenoids pathway to facilitate genetic and metabolic engineering studies. The obtained three-dimensional arrangement of the amino acids revealed the regional structures and folding of the diverse segments of helices, sheets, turns. This information is a key point to unveil the protein's operation mechanism. Besides, we confirmed the suitability of bioinformatic approaches for analysing the gene structures and identifying the new Psy genes in unstudied microalgal strains.

Butyryl/Caproyl-CoA:Acetate CoA-Transferase: Cloning, Expression and Characterization of the Key Enzyme Involved in Medium-Chain Fatty Acid Biosynthesis

Coenzyme A transferases (CoATs) are important enzymes involved in carbon chain elongation contributing to medium-chain fatty acid (MCFA) biosynthesis. For example, butyryl-CoA:acetate CoA transferase (BCoAT) is responsible for the final step of butyrate synthesis from butyryl-CoA. However, little is known about caproyl-CoA:acetate CoA-transferase (CCoAT), which is responsible for the final step of caproate synthesis from caproyl-CoA. In this study, two CoAT genes from Ruminococcaceae bacterium CPB6 and Clostridium tyrobutyricum BEY8 were identified by gene cloning and expression analysis. The enzyme assays and kinetic studies were carried out using butyryl-CoA or caproyl-CoA as the substrate. CPB6-CoAT can catalyze the conversion of both butyryl-CoA to butyrate and caproyl-CoA to caproate, but its catalytic efficiency with caproyl-CoA as the substrate was 3.8 times higher than that with butyryl-CoA. In contrast, BEY8-CoAT had only BCoAT activity, not CCoAT activity. This demonstrated the existence of a specific CCoAT involved in chain elongation via the reverse β-oxidation pathway. Comparative bioinformatics analysis showed the presence of a highly conserved motif (GGQXDFXXGAXX) in CoATs, which is predicted to be the active center of CoATs. Single point mutations in the conserved motif of CPB6-CoAT (Asp346 and Ala351) led to marked decreases in the activity for butyryl-CoA and caproyl-CoA, indicating that the conserved motif is the active center of CPB6-CoAT, and sites Asp346 and Ala351 were critical residues that affect enzymatic activity. This work provides insight into the function of CCoAT in caproic acid biosynthesis and improves the understanding of the chain elongation pathway for MCFA production.

An evolutionary non-conserved motif in Helicobacter pylori arginase mediates positioning of the loop containing the catalytic residue for catalysis

The binuclear metalloenzyme Helicobacter pylori arginase is important for pathogenesis of the bacterium in the human stomach. Despite conservation of the catalytic residues, this single Trp enzyme has an insertion sequence (–153ESEEKAWQKLCSL165–) that is extremely crucial to function. This sequence contains the critical residues, which are conserved in the homolog of other Helicobacter gastric pathogens. However, the underlying basis for the role of this motif in catalytic function is not completely understood. Here, we used biochemical, biophysical and molecular dynamics simulations studies to determine that Glu155 of this stretch interacts with both Lys57 and Ser152. These interactions are essential for positioning of the motif through Trp159, which is located near Glu155 (His122–Trp159–Tyr125 contact is essential to tertiary structural integrity). The individual or double mutation of Lys57 and Ser152 to Ala considerably reduces catalytic activity with Lys57 to Ala being more significant, indicating they are crucial to function. Our data suggest that the Lys57–Glu155–Ser152 interaction influences the positioning of the loop containing the catalytic His133 so that this His can participate in catalysis, thereby providing a mechanistic understanding into the role of this motif in catalytic function. Lys57 was also found only in the arginases of other Helicobacter gastric pathogens. Based on the non-conserved motif, we found a new molecule, which specifically inhibits this enzyme. Thus, the present study not only provides a molecular basis into the role of this motif in function, but also offers an opportunity for the design of inhibitors with greater efficacy.

From Data Mining of Chitinophaga sp. Genome to Enzyme Discovery of a Hyperthermophilic Metallocarboxypeptidase

For several centuries, microorganisms and enzymes have been used for many different applications. Although many enzymes with industrial applications have already been reported, different screening technologies, methods and approaches are constantly being developed in order to allow the identification of enzymes with even more interesting applications. In our work, we have performed data mining on the Chitinophaga sp. genome, a gram-negative bacterium isolated from a bacterial consortium of sugarcane bagasse isolated from an ethanol plant. The analysis of 8 Mb allowed the identification of the chtcp gene, previously annotated as putative Cht4039. The corresponding codified enzyme, denominated as ChtCP, showed the HEXXH conserved motif of family M32 from thermostable carboxypeptidases. After expression in E. coli, the recombinant enzyme was characterized biochemically. ChtCP showed the highest activity versus benziloxicarbonil Ala-Trp at pH 7.5, suggesting a preference for hydrophobic substrates. Surprisingly, the highest activity of ChtCP observed was between 55 °C and 75 °C, and 62% activity was still displayed at 100 °C. We observed that Ca2+, Ba2+, Mn2+ and Mg2+ ions had a positive effect on the activity of ChtCP, and an increase of 30 °C in the melting temperature was observed in the presence of Co2+. These features together with the structure of ChtCP at 1.2 Å highlight the relevance of ChtCP for further biotechnological applications.