A Unique Sulfotransferase-Involving Strigolactone Biosynthetic Route in Sorghum

LOW GERMINATION STIMULANT 1 (LGS1) plays an important role in strigolactones (SLs) biosynthesis and Striga resistance in sorghum, but the catalytic function remains unclear. Using the recently developed SL-producing microbial consortia, we examined the activities of sorghum MORE AXILLARY GROWTH1 (MAX1) analogs and LGS1. Surprisingly, SbMAX1a (cytochrome P450 711A enzyme in sorghum) synthesized 18-hydroxy-carlactonoic acid (18-hydroxy-CLA) directly from carlactone (CL) through four-step oxidations. The further oxidated product orobanchol (OB) was also detected in the microbial consortium. Further addition of LGS1 led to the synthesis of both 5-deoxystrigol (5DS) and 4-deoxyorobanchol (4DO). Further biochemical characterization found that LGS1 functions after SbMAX1a by converting 18-hydroxy-CLA to 5DS and 4DO possibly through a sulfonation-mediated pathway. The unique functions of SbMAX1 and LGS1 imply a previously unknown synthetic route toward SLs.

A Structural Approach to Anti-Virulence: A Discovery Pipeline

The anti-virulence strategy is designed to prevent bacterial virulence factors produced by pathogenic bacteria from initiating and sustaining an infection. One family of bacterial virulence factors is the mono-ADP-ribosyltransferase toxins, which are produced by pathogens as tools to compromise the target host cell. These toxins are bacterial enzymes that exploit host cellular NAD+ as the donor substrate to modify an essential macromolecule acceptor target in the host cell. This biochemical reaction modifies the target macromolecule (often protein or DNA) and functions in a binary fashion to turn the target activity on or off by blocking or impairing a critical process or pathway in the host. A structural biology approach to the anti-virulence method to neutralize the cytotoxic effect of these factors requires the search and design of small molecules that bind tightly to the enzyme active site and prevent catalytic function essentially disarming the pathogen. This method requires a high-resolution structure to serve as the model for small molecule inhibitor development, which illuminates the path to drug development. This alternative strategy to antibiotic therapy represents a paradigm shift that may circumvent multi-drug resistance in the offending microbe through anti-virulence therapy. In this report, the rationale for the anti-virulence structural approach will be discussed along with recent efforts to apply this method to treat honey bee diseases using natural products.

Human Group IIA Phospholipase A2—Three Decades on from Its Discovery

Phospholipase A2 (PLA2) enzymes were first recognized as an enzyme activity class in 1961. The secreted (sPLA2) enzymes were the first of the five major classes of human PLA2s to be identified and now number nine catalytically-active structurally homologous proteins. The best-studied of these, group IIA sPLA2, has a clear role in the physiological response to infection and minor injury and acts as an amplifier of pathological inflammation. The enzyme has been a target for anti-inflammatory drug development in multiple disorders where chronic inflammation is a driver of pathology since its cloning in 1989. Despite intensive effort, no clinically approved medicines targeting the enzyme activity have yet been developed. This review catalogues the major discoveries in the human group IIA sPLA2 field, focusing on features of enzyme function that may explain this lack of success and discusses future research that may assist in realizing the potential benefit of targeting this enzyme. Functionally-selective inhibitors together with isoform-selective inhibitors are necessary to limit the apparent toxicity of previous drugs. There is also a need to define the relevance of the catalytic function of hGIIA to human inflammatory pathology relative to its recently-discovered catalysis-independent function.

PTEN differentially regulates endocytosis, migration, and proliferation in the enteric protozoan parasite Entamoeba histolytica

PTEN is a lipid phosphatase that is highly conserved and involved in a broad range of biological processes includingcytoskeletal reorganization, endocytosis, signal transduction, and cell migration in all eukaryotes. Although regulation of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] signaling via PTEN has been well established in model organisms and mammals, it remains elusive in the parasitic protist E. histolytica, which heavily relies on PtdIns phosphate(s)-dependent membrane traffic, migration, and phago- and trogocytosis for its pathogenesis. In this study, we characterized the major PTEN from E. histolytica, EhPTEN1, which shows the highest expression at the transcript level in the trophozoite stage among 6 possible PTENs, to understand the significance of PtdIns(3,4,5)P3 signaling in this parasite. Live imaging of GFP-EhPTEN1 expressing amebic trophozoites showed localization mainly in the cytosol with a higher concentration at pseudopods and the extending edge of the phago- and trogocytic cups. Furthermore, quantitative analysis of phago- and trogocytosis using a confocal image cytometer showed that overexpression of EhPTEN1 caused reduction in trogo- and phagocytosis while transcriptional gene silencing of EhPTEN1 gene caused opposite phenotypes. These data suggest that EhPTEN1 has an inhibitory role in these biological processes. Conversely, EhPTEN1 acts as a positive regulator for fluid-phase and receptor-mediated endocytosis in E. histolytica trophozoites. Moreover, we showed that EhPTEN1 was required for optimal growth and migration of this parasite. Finally, the phosphatase activity of EhPTEN1 towards PtdIns(3,4,5)P3 was demonstrated, suggesting that the biological roles of EhPTEN1 are likely linked to its catalytic function. Taken together, these results indicate that EhPTEN1 differentially regulates multiple cellular activities essential for proliferation and pathogenesis of the organism, via PtdIns(3,4,5)P3 signaling. Elucidation of biological roles of PTEN and PtdIns(3,4,5)P3 signaling at the molecular levels promotes our understanding of the pathogenesis of this parasite and potentially leads to the design of novel therapeutics against amebiasis.

Particition of Sodium-potassium Adenosine Triphosphatases in Homeostasis Regulation

A literature review presented an analysis of data regarding the mechanisms of the Na pump in nephron and hormonal regulators of enzyme activity, including enzymatic catalysts. Investigating the regulatory mechanisms of metabolic processes can facilitate the development of new strategies to repair various pathological conditions. Among these functional proteins, Na+/K+ATPase is responsible for the regulation of hydroionic homeostasis and signaling. Ion transport in different parts of the nephron is mediated via sodium transporters, which are characterized by a clear topographical expression. In the oligomeric Na+/K+ATPase molecule, the α-subunit comprises 10 transmembrane domains and performs a catalytic function. The signal function of Na+/K+ATPase and its interaction with the molecular environment in lipid microdomains involve rafts and caveolae. Analysis of the literature data demonstrated an important function of Na+/K+ATPase, along with its interaction with caveolin-1, in the regulation of intracellular cholesterol traffic. Moreover, reciprocal interactions of enzymes and cholesterol have been indicated. The status of Na+/K+ATPase activity is affected by hypoxia, reactive oxygen species, lipid peroxidation (LPO), increased cholesterol concentrations, and the viscosity of the cytoplasmic membrane. Ecological pollutants, including heavy metals, have significant effects on enzyme activity in nephron, hepatocytes and cardiomyocytes. Thus, available literature data indicate an important role of Na+/K+ATPase in the regulation of metabolic processes.

Conserved Amino Acid Moieties of Candidatus Desulforudis audaxviator MazF Determine Ribonuclease Activity and Specificity

The toxin-antitoxin (TA) system, inherent to various prokaryotes, plays a critical role in survival and adaptation to diverse environmental stresses. The toxin MazF, belonging to the type II TA system, functions as a sequence-specific ribonuclease that recognizes 3 to 7 bases. In recent studies, crystallographic analysis of MazFs from several species have suggested the presence of amino acid sites important for MazF substrate RNA binding and for its catalytic activity. Herein, we characterized MazF obtained from Candidatus Desulforudis audaxviator (MazF-Da) and identified the amino acid residues necessary for its catalytic function. MazF-Da, expressed using a cell-free protein synthesis system, is a six-base-recognition-specific ribonuclease that preferentially cleaves UACAAA sequences and weakly cleaves UACGAA and UACUAA sequences. We found that MazF-Da exhibited the highest activity at around 60°C. Analysis using mutants with a single mutation at an amino acid residue site that is well conserved across various MazF toxins showed that G18, E20, R25, and P26 were important for the ribonuclease activity of MazF-Da. The recognition sequence of the N36A mutant differed from that of the wild type. This mutant cleaved UACAAG sequences in addition to UACAAA sequences, but did not cleave UACGAA or UACUAA sequences, suggesting that Asn36 affects the loosening and narrowing of MazF-Da cleavage sequence recognition. Our study posits UACAAA as the recognition sequence of MazF-Da and provides insight into the amino acid sites that are key to its unique enzymatic properties.

Comparative Modeling And Enzymatic Affinity of Novel Haloacid Dehalogenase From Bacillus Megaterium Strain BHS1 Isolated From Alkaline Blue Lake In Turkey

This is the first structural model of L-haloacid dehalogenase (DehLBHS1) isolated from alkalotolerant Bacillus megaterium BHS1, which has been known to degrading halogenated environmental contaminants. The study suggested five important key amino acid residues of DehLBHS1, namely Arg40, Phe59, Asn118, Asn176 and Trp178 important for catalysis and molecular recognition of haloalkanoic acid. Alkatolerant DehLBHS1was modeled by I-TASSER with the best C-score 1.23. Model validation was carried out utilising PROCHECK to produce the Ramachandran map with 89.2 percent of its residues were found in the most preferred region, indicating that the model was appropriate. The Molecular docking (MD) simulation found that the DehLBHS1 preferred 2,2DCP more than other substrates and formed one hydrogen bond with Arg40 and minimum energy -2.5 kJ/ mol. Molecular dynamics has verified the substrate preference towards 2,2DCP based on RMSD, RMSF, Gyration, Hydrogen bond and Molecular distance. This structural knowledge from DehLBHS1 structural perspective gives insights into substrate specificity and catalytic function to exploit DehLBHS1 of BHS1 strain in degrading 2,2-DCP in the polluted alkaline environments.