Improved, reference quality genome sequence of the plastic-degrading greater wax moth, Galleria mellonella.

Galleria mellonella is a pest of honeybees in many countries because its larvae feed on beeswax. However, G. mellonella larvae can also eat various plastics, including polyethylene, polystyrene and polypropylene, so the species is garnering increasing interest as a tool for plastic biodegradation research. This paper presents an improved genome (99.3% completed lepidoptera_odb10 BUSCO; genome mode) for G. mellonella. This 472 Mb genome is in 221 contigs with an N50 of 6.4 MB and contains 13,604 protein-coding genes. Genes that code for known and putative polyethylene-degrading enzymes and their similarity to proteins found in other Lepidoptera are highlighted. An analysis of secretory proteins more likely to be involved in the plastic catabolic process has also been carried out.

Metabolic disruptions and impaired reproductive fitness in wild-caught freshwater turtles (Emydura macquarii macquarii) exposed to elevated per- and polyfluoroalkyl substances (PFAS).

Per- and poly-fluoroalkyl substances (PFAS) pose a threat to organisms and ecosystems due to their persistent nature. Ecotoxicology endpoints used in regulatory guidelines may not reflect multiple, low-level but persistent stressors. This study examines the biological effects of PFAS on Eastern short-necked turtles in Queensland, Australia. In this study, blood samples were collected and analysed for PFAS, hormone levels, and functional omics endpoints. High levels of PFAS were found in turtles at the impacted site, with PFOS being the dominant constituent. The PFAS profiles of males and females differed, with males having higher PFAS concentrations. Hormone concentrations differed between impacted and reference sites in male turtles, with elevated testosterone and corticosterone indicative of stress. Further, energy utilisation, nucleotide synthesis, nitrogen metabolism, and amino acid synthesis were altered in both male and female turtles from PFAS-impacted sites. Both sexes show similar metabolic responses to environmental stressors from the PFAS-contaminated site, which may adversely affect their reproductive fitness. Purine metabolism, caffeine metabolism, and ferroptosis pathway changes in turtles can cause gout, cell death, and overall health problems. Further, the study showed that prolonged exposure to elevated PFAS levels in the wild could compromise turtle reproductive fitness by disrupting reproductive steroids and metabolic pathways.

Patterns of Variation in the Usage of Fatty Acid Chains among Classes of Ester and Ether Neutral Lipids and Phospholipids in the Queensland Fruit Fly.

Modern lipidomics has the power and sensitivity to elucidate the role of insects' lipidomes in their adaptations to the environment at a mechanistic molecular level. However, few lipidomic studies have yet been conducted on insects beyond model species such as Drosophila melanogaster. Here, we present the lipidome of adult males of another higher dipteran frugivore, Bactrocera tryoni. We describe 421 lipids across 15 classes of ester neutral lipids and phospholipids and ether neutral lipids and phospholipids. Most of the lipids are specified in terms of the carbon and double bond contents of each constituent hydrocarbon chain, and more ether lipids are specified to this degree than in any previous insect lipidomic analyses. Class-specific profiles of chain length and (un)saturation are broadly similar to those reported in D. melanogaster, although we found fewer medium-length chains in ether lipids. The high level of chain specification in our dataset also revealed widespread non-random combinations of different chain types in several ester lipid classes, including deficits of combinations involving chains of the same carbon and double bond contents among four phospholipid classes and excesses of combinations of dissimilar chains in several classes. Large differences were also found in the length and double bond profiles of the acyl vs. alkyl or alkenyl chains of the ether lipids. Work on other organisms suggests some of the differences observed will be functionally consequential and mediated, at least in part, by differences in substrate specificity among enzymes in lipid synthesis and remodelling pathways. Interrogation of the B. tryoni genome showed it has comparable levels of diversity overall in these enzymes but with some gene gain/loss differences and considerable sequence divergence from D. melanogaster.

Wide substrate range for a candidate bioremediation enzyme isolated from Nocardioides sp. strain SG-4 G.

Narrow substrate ranges can impact heavily on the range of applications and hence commercial viability of candidate bioremediation enzymes. Here we show that an ester hydrolase from Nocardioides strain SG-4 G has potential as a bioremediation agent against various pollutants that can be detoxified by hydrolytic cleavage of some carboxylester, carbamate, or amide linkages. Previously we showed that a radiation-killed, freeze-dried preparation (ZimA) of this strain can rapidly degrade the benzimidazole fungicide carbendazim due to the activity of a specific ester hydrolase, MheI. Here, we report that ZimA also has substantial hydrolytic activity against phthalate diesters (dimethyl, dibutyl, and dioctyl phthalate), anilide (propanil and monalide), and carbamate ester (chlorpropham) herbicides under laboratory conditions. The reaction products are substantially less toxic, or inactive as herbicides, than the parent compounds. Tests of strain SG-4 G and Escherichia coli expressing MheI found they were also able to hydrolyse dimethyl phthalate, propanil, and chlorpropham, indicating that MheI is principally responsible for the above activities.

Genetic variation for rectal gland volatiles among recently collected isofemale lines and a domesticated strain of Queensland fruit fly, Bactrocera tryoni (Diptera: Tephritidae).

Divergence between populations in mating behaviour can function as a potent premating isolating mechanism and promote speciation. However, very few cases of inherited intraspecific variation in sexual signalling have been reported in tephritid fruit flies, despite them being a highly speciose family. We tested for such variation in one tephritid, the Queensland fruit fly, Bactrocera tryoni (Qfly). Qfly mating behaviour depends on volatiles secreted from male rectal glands but no role for the volatiles from female rectal glands has yet been reported. We previously detected over 100 volatile compounds in male rectal glands and identified over 30 of them. Similar numbers were recorded in females. However, many compounds showed presence/absence differences between the sexes and many others showed quantitative differences between them. Here we report inherited variation among 24 Qfly lines (23 isofemale lines established from recent field collections and one domesticated line) in the abundance of three esters, two alcohols, two amides, an aldehyde and 18 unidentified volatiles in male rectal glands. We did not find any compounds in female rectal glands that varied significantly among the lines, although this may at least partly reflect lower female sample numbers. Most of the 26 male compounds that differed between lines were more abundant in the domesticated line than any of the recently established isofemale lines, which concurs with other evidence for changes in mating behaviour during domestication of this species. There were also large differences in several of the 26 compounds among the isofemale lines, and some of these differences were associated with the regions from which the lines were collected. While some of the variation in different compounds was correlated across lines, much of it was not, implicating involvement of multiple genes. Our findings parallel reports of geographic variation in other Qfly traits and point to inherited differences in reproductive physiology that could provide a basis for evolution of premating isolation between ecotypes.

Rounding up the annual ryegrass genome: High-quality reference genome of Lolium rigidum.

The genome of the major agricultural weed species, annual ryegrass (Lolium rigidum) was assembled, annotated and analysed. Annual ryegrass is a major weed in grain cropping, and has the remarkable capacity to evolve resistance to herbicides with various modes of action. The chromosome-level assembly was achieved using short- and long-read sequencing in combination with Hi-C mapping. The assembly size is 2.44 Gb with N50 = 361.79 Mb across 1,764 scaffolds where the seven longest sequences correspond to the seven chromosomes. Genome completeness assessed through BUSCO returned a 99.8% score for complete (unique and duplicated) and fragmented genes using the Viridiplantae set. We found evidence for the expansion of herbicide resistance-related gene families including detoxification genes. The reference genome of L. rigidum is a critical asset for leveraging genetic information for the management of this highly problematic weed species.

Diversity and sex differences in rectal gland volatiles of Queensland fruit fly, Bactrocera tryoni (Diptera: Tephritidae).

Rectal gland volatiles are key mediators of sexual interactions in tephritid fruit flies. We used solid-phase microextraction (SPME) plus gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detection (GC-FID) to substantially expand rectal gland chemical characterisation of the Queensland fruit fly (Bactrocera tryoni (Diptera: Tephritidae); Qfly). The SPME GC-MS analysis identified 24 of the 30 compounds previously recorded from Qfly rectal glands, plus another 21 compounds that had not previously been reported. A few amides and fatty acid esters dominated the chromatograms of males and females respectively, but we also found other esters, alcohols and aldehydes and a ketone. The GC-FID analyses also revealed over 150 others, as yet unidentified, volatiles, generally in lesser amounts. The GC-FID analyses also showed 49 and 12 compounds were male- and female-specific, respectively, both in single sex (virgin) and mixed sex (mostly mated) groups. Another ten compounds were male-specific among virgins but undetected in mixed sex groups, and 29 were undetected in virgins but male-specific in mixed sex groups. The corresponding figures for females were four and zero, respectively. Most short retention time peaks (including a ketone and an ester) were male-specific, whereas most female-biased peaks (including five fatty acid esters) had long retention times. Our results indicate previously unsuspected diversity of rectal gland volatiles that might have pheromone functions in males, but far fewer in females.

Population differences and domestication effects on mating and remating frequencies in Queensland fruit fly.

Females of many insect species are unreceptive to remating for a period following their first mating. This inhibitory effect may be mediated by either the female or her first mate, or both, and often reflects the complex interplay of reproductive strategies between the sexes. Natural variation in remating inhibition and how this phenotype responds to captive breeding are largely unexplored in insects, including many pest species. We investigated genetic variation in remating propensity in the Queensland fruit fly, Bactrocera tryoni, using strains differing in source locality and degree of domestication. We found up to threefold inherited variation between strains from different localities in the level of intra-strain remating inhibition. The level of inhibition also declined significantly during domestication, which implied the existence of genetic variation for this trait within the starting populations as well. Inter-strain mating and remating trials showed that the strain differences were mainly due to the genotypes of the female and, to a lesser extent, the second male, with little effect of the initial male genotype. Implications for our understanding of fruit fly reproductive biology and population genetics and the design of Sterile Insect Technique pest management programs are discussed.

Antiradical Properties of trans-2-(4-substituted-styryl)-thiophene.

2-substituted thiophene compounds with electron donating and electron withdrawing p-phenyl substitution were synthesized and studied their radical scavenging properties using DPPH assay and DFT method. It is shown that p-hydroxy and p-amino phenyl substituted compound exhibit radical scavenging activity. From DFT and radical scavenging studies, a correlation between IC50 with the bond dissociation enthalpy, proton affinity, ground state dipole moment and optical band gap of compound is found. Compounds 1-3 with electron withdrawing substituent (NO2, CN, Cl) do not show any radical scavenging properties, whereas compounds 6-7 with electron donating substituent (OH, NH2) show antiradical properties. Further, the antiradical activity is reduced drastically by replacing the -OH and -NH2 with methoxy and -N-alkylating group respectively in 6 and 7. The compound with p-hydroxy phenyl substitution, exhibits stronger antiradical activity as compared to the p-amino phenyl substitution due to smaller O-H bond dissociation energy as compared to the N-H bond. From DPPH and DFT studies, it is suggested that the radical scavenging activity in 2-substituted thiophene is occurred through proton transfer mechanism. The other possible SET, SPLET mechanisms are also corroborated. Graphical Abstract Antiradical properties of trans-2-(4-substituted-styryl)-thiophene Anamika Gusain, Naresh Kumar, Jagdeep Kumar, Gunjan Pandey, Prasanta Kumar Hota.

Cuticular Chemistry of the Queensland Fruit Fly Bactrocera tryoni (Froggatt).

The cuticular layer of the insect exoskeleton contains diverse compounds that serve important biological functions, including the maintenance of homeostasis by protecting against water loss, protection from injury, pathogens and insecticides, and communication. Bactrocera tryoni (Froggatt) is the most destructive pest of fruit production in Australia, yet there are no published accounts of this species' cuticular chemistry. We here provide a comprehensive description of B. tryoni cuticular chemistry. We used gas chromatography-mass spectrometry to identify and characterize compounds in hexane extracts of B. tryoni adults reared from larvae in naturally infested fruits. The compounds found included spiroacetals, aliphatic amides, saturated/unsaturated and methyl branched C12 to C20 chain esters and C29 to C33 normal and methyl-branched alkanes. The spiroacetals and esters were found to be specific to mature females, while the amides were found in both sexes. Normal and methyl-branched alkanes were qualitatively the same in all age and sex groups but some of the alkanes differed in amounts (as estimated from internal standard-normalized peak areas) between mature males and females, as well as between mature and immature flies. This study provides essential foundations for studies investigating the functions of cuticular chemistry in this economically important species.

A Multiparametric Assay Platform for Simultaneous In Vivo Assessment of Pronephric Morphology, Renal Function and Heart Rate in Larval Zebrafish.

Automated high-throughput workflows allow for chemical toxicity testing and drug discovery in zebrafish disease models. Due to its conserved structural and functional properties, the zebrafish pronephros offers a unique model to study renal development and disease at larger scale. Ideally, scoring of pronephric phenotypes includes morphological and functional assessments within the same larva. However, to efficiently upscale such assays, refinement of existing methods is required. Here, we describe the development of a multiparametric in vivo screening pipeline for parallel assessment of pronephric morphology, kidney function and heart rate within the same larva on a single imaging platform. To this end, we developed a novel 3D-printed orientation tool enabling multiple consistent orientations of larvae in agarose-filled microplates. Dorsal pronephros imaging was followed by assessing renal clearance and heart rates upon fluorescein isothiocyanate (FITC)-inulin microinjection using automated time-lapse imaging of laterally positioned larvae. The pipeline was benchmarked using a set of drugs known to induce developmental nephrotoxicity in humans and zebrafish. Drug-induced reductions in renal clearance and heart rate alterations were detected even in larvae exhibiting minor pronephric phenotypes. In conclusion, the developed workflow enables rapid and semi-automated in vivo assessment of multiple morphological and functional parameters.

Oxidative Catabolism of (+)-Pinoresinol Is Initiated by an Unusual Flavocytochrome Encoded by Translationally Coupled Genes within a Cluster of (+)-Pinoresinol-Coinduced Genes in Pseudomonas sp. Strain SG-MS2.

Burkholderia sp. strain SG-MS1 and Pseudomonas sp. strain SG-MS2 have previously been found to mineralize (+)-pinoresinol through a common catabolic pathway. Here, we used comparative genomics, proteomics, protein semipurification, and heterologous expression to identify a flavoprotein from the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family in SG-MS2 that carries out the initial hydroxylation of (+)-pinoresinol at the benzylic carbon. The cognate gene is translationally coupled with a downstream cytochrome gene, and the cytochrome is required for activity. The flavoprotein has a unique combination of cofactor binding and cytochrome requirements for the VAO/PCMH family. The heterologously expressed enzyme has a Km of 1.17 µM for (+)-pinoresinol. The enzyme is overexpressed in strain SG-MS2 upon exposure to (+)-pinoresinol, along with 45 other proteins, 22 of which were found to be encoded by genes in an approximately 35.1-kb cluster also containing the flavoprotein and cytochrome genes. Homologs of 18 of these 22 genes, plus the flavoprotein and cytochrome genes, were also found in a 38.7-kb cluster in SG-MS1. The amino acid identities of four of the other proteins within the SG-MS2 cluster suggest they catalyze conversion of hydroxylated pinoresinol to protocatechuate and 2-methoxyhydroquinone. Nine other proteins upregulated in SG-MS2 on exposure to (+)-pinoresinol appear to be homologs of proteins known to comprise the protocatechuate and 2-methoxyhydroquinone catabolic pathways, but only three of the cognate genes lie within the cluster containing the flavoprotein and cytochrome genes.IMPORTANCE (+)-Pinoresinol is an important plant defense compound, a major food lignan for humans and some other animals, and the model compound used to study degradation of the ß-ß' linkages in lignin. We report a gene cluster, in one strain each of Pseudomonas and Burkholderia, that is involved in the oxidative catabolism of (+)-pinoresinol. The flavoprotein component of the α-hydroxylase which heads the pathway belongs to the 4-phenol oxidizing (4PO) subgroup of the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family but constitutes a novel combination of cofactor and electron acceptor properties for the family. It is translationally coupled with a cytochrome gene whose product is also required for activity. The work casts new light on the biology of (+)-pinoresinol and its transformation to other bioactive molecules. Potential applications of the findings include new options for deconstructing lignin into useful chemicals and the generation of new phytoestrogenic enterolactones from lignans.

A Smart Imaging Workflow for Organ-Specific Screening in a Cystic Kidney Zebrafish Disease Model.

The zebrafish is being increasingly used in biomedical research and drug discovery to conduct large-scale compound screening. However, there is a lack of accessible methodologies to enable automated imaging and scoring of tissue-specific phenotypes at enhanced resolution. Here, we present the development of an automated imaging pipeline to identify chemical modifiers of glomerular cyst formation in a zebrafish model for human cystic kidney disease. Morpholino-mediated knockdown of intraflagellar transport protein Ift172 in Tg(wt1b:EGFP) embryos was used to induce large glomerular cysts representing a robustly scorable phenotypic readout. Compound-treated embryos were consistently aligned within the cavities of agarose-filled microplates. By interfacing feature detection algorithms with automated microscopy, a smart imaging workflow for detection, centring and zooming in on regions of interests was established, which enabled the automated capturing of standardised higher resolution datasets of pronephric areas. High-content screening datasets were processed and analysed using custom-developed heuristic algorithms implemented in common open-source image analysis software. The workflow enables highly efficient profiling of entire compound libraries and scoring of kidney-specific morphological phenotypes in thousands of zebrafish embryos. The demonstrated toolset covers all the aspects of a complex whole organism screening assay and can be adapted to other organs, specimens or applications.

The molecular basis for the neofunctionalization of the juvenile hormone esterase duplication in Drosophila.

The Drosophila melanogaster enzymes juvenile hormone esterase (DmJHE) and its duplicate, DmJHEdup, present ideal examples for studying the structural changes involved in the neofunctionalization of enzyme duplicates. DmJHE is a hormone esterase with precise regulation and highly specific activity for its substrate, juvenile hormone. DmJHEdup is an odorant degrading esterase (ODE) responsible for processing various kairomones in antennae. Our phylogenetic analysis shows that the JHE lineage predates the hemi/holometabolan split and that several duplications of JHEs have been templates for the evolution of secreted ß-esterases such as ODEs through the course of insect evolution. Our biochemical comparisons further show that DmJHE has sufficient substrate promiscuity and activity against odorant esters for a duplicate to evolve a general ODE function against a range of mid-long chain food esters, as is shown in DmJHEdup. This substrate range complements that of the only other general ODE known in this species, Esterase 6. Homology models of DmJHE and DmJHEdup enabled comparisons between each enzyme and the known structures of a lepidopteran JHE and Esterase 6. Both JHEs showed very similar active sites despite low sequence identity (30%). Both ODEs differed drastically from the JHEs and each other, explaining their complementary substrate ranges. A small number of amino acid changes are identified that may have been involved in the early stages of the neofunctionalization of DmJHEdup. Our results provide key insights into the process of neofunctionalization and the structural changes that can be involved.

Zebrafish as a Model for Drug Screening in Genetic Kidney Diseases.

Genetic disorders account for a wide range of renal diseases emerging during childhood and adolescence. Due to the utilization of modern biochemical and biomedical techniques, the number of identified disease-associated genes is increasing rapidly. Modeling of congenital human disease in animals is key to our understanding of the biological mechanism underlying pathological processes and thus developing novel potential treatment options. The zebrafish (Danio rerio) has been established as a versatile small vertebrate organism that is widely used for studying human inherited diseases. Genetic accessibility in combination with elegant experimental methods in zebrafish permit modeling of human genetic diseases and dissecting the perturbation of underlying cellular networks and physiological processes. Beyond its utility for genetic analysis and pathophysiological and mechanistic studies, zebrafish embryos, and larvae are amenable for phenotypic screening approaches employing high-content and high-throughput experiments using automated microscopy. This includes large-scale chemical screening experiments using genetic models for searching for disease-modulating compounds. Phenotype-based approaches of drug discovery have been successfully performed in diverse zebrafish-based screening applications with various phenotypic readouts. As a result, these can lead to the identification of candidate substances that are further examined in preclinical and clinical trials. In this review, we discuss zebrafish models for inherited kidney disease as well as requirements and considerations for the technical realization of drug screening experiments in zebrafish.

Isolation of the (+)-Pinoresinol-Mineralizing Pseudomonas sp. Strain SG-MS2 and Elucidation of Its Catabolic Pathway.

Pinoresinol is a dimer of two ß-ß'-linked coniferyl alcohol molecules. It is both a plant defense molecule synthesized through the shikimic acid pathway and a representative of several ß-ß-linked dimers produced during the microbial degradation of lignin in dead plant material. Until now, little has been known about the bacterial catabolism of such dimers. Here we report the isolation of the efficient (+)-pinoresinol-mineralizing Pseudomonas sp. strain SG-MS2 and its catabolic pathway. Degradation of pinoresinol in this strain is inducible and proceeds via a novel oxidative route, which is in contrast to the previously reported reductive transformation by other bacteria. Based on enzyme assays and bacterial growth, cell suspension, and resting cell studies, we provide conclusive evidence that pinoresinol degradation in strain SG-MS2 is initiated by benzylic hydroxylation, generating a hemiketal via a quinone methide intermediate, which is then hydrated at the benzylic carbon by water. The hemiketal, which stays in equilibrium with the corresponding keto alcohol, undergoes an aryl-alkyl cleavage to generate a lactone and 2-methoxyhydroquinone. While the fate of 2-methoxyhydroquinone is not investigated further, it is assumed to be assimilated by ring cleavage. The lactone is further metabolized via two routes, namely, lactone ring cleavage and benzylic hydroxylation via a quinone methide intermediate, as described above. The resulting hemiketal again exists in equilibrium with a keto alcohol. Our evidence suggests that both routes of lactone metabolism lead to vanillin and vanillic acid, which we show can then be mineralized by strain SG-MS2.IMPORTANCE The oxidative catabolism of (+)-pinoresinol degradation elucidated here is fundamentally different from the reductive cometabolism reported for two previously characterized bacteria. Our findings open up new opportunities to use lignin for the biosynthesis of vanillin, a key flavoring agent in foods, beverages, and pharmaceuticals, as well as various new lactones. Our work also has implications for the study of new pinoresinol metabolites in human health. The enterodiol and enterolactone produced through reductive transformation of pinoresinol by gut microbes have already been associated with decreased risks of cancer and cardiovascular diseases. The metabolites from oxidative metabolism we find here also deserve attention in this respect.

3-Aminopropyltrimethoxysilane mediated solvent induced synthesis of gold nanoparticles for biomedical applications.

The effect of acetone on 3-aminopropyltrimethoxysilane (3-APTMS) mediated synthesis of gold nanoparticles (AuNPs) in polar protic, polar aprotic and non polar solvents specifically water, acetone and chloroform has been studied. The findings revealed that acetone promotes the formation of siloxane polymer in chloroform and acetone solvents, but not in water, while AuNPs are formed in all cases. The bifunctional nature of 3-APTMS [NH2(CH2)3Si(OMe)3] controls the formation of siloxane polymer through hydrolysis and condensation of trimethoxysilyl group whereas the amine moieties are likely to form imine linkage, which are involved in the synthesis of gold nanoparticles. The siloxane polymer with imine linkage can be formed both in absence and the presence of gold precursor (i.e. HAuCl4). Accordingly, siloxane-gold nanoparticles can be made in different morphology, through control over the process, in three ways; (i) all precursors (Au3+, 3-APTMS and acetone) mixed simultaneously yielding [(siloxane-Ausim)], (ii) polymer made first followed by sequential reduction of Au3+ in homogenous suspension forming (Au-siloxanehomo)seq and (iii) polymer made into thin film followed by sequential reduction of Au3+ in the heterogeneous system yielding (Au-siloxanehetero)seq. The AuNPs produced with the use of water as solvent is different from the three described above as discrete spherical AuNPs are formed as [(AuNPs)water] and in this case siloxane polymer is not formed. The results based on AFM and SEM characterizations revealed porous morphology of NPs fabricated in chloroform/acetone, due to the formation of siloxane polymer, and non-porous morphology for the same in water. The "(Au-siloxanehetero)seq" efficiently promote the reduction of p-nitrophenol with good operational stability, justifying its interest for heterogeneous catalysis. On the other hand, (siloxane-Ausim) and (Au-siloxanehomo)seq can be also deposited onto glassy carbon surfaces to yield polymer modified electrodes for subsequent electroanalytical applications. The cyclic voltammetry response of such modified electrodes to a redox probe (i.e., Fe(CN)63-/4-) was characteristic of diffusion controlled and thin-layer electrochemical behaviour respectively as a function of non-porous and porous nature of the polymer. The as made nanomaterial shows excellent biocatalyst for dopamine sensing based on peroxidase mimetic activity. The typical result on dopamine sensing is reported.

Mycobacterial F420H2-Dependent Reductases Promiscuously Reduce Diverse Compounds through a Common Mechanism.

An unusual aspect of actinobacterial metabolism is the use of the redox cofactor F420. Studies have shown that actinobacterial F420H2-dependent reductases promiscuously hydrogenate diverse organic compounds in biodegradative and biosynthetic processes. These enzymes therefore represent promising candidates for next-generation industrial biocatalysts. In this work, we undertook the first broad survey of these enzymes as potential industrial biocatalysts by exploring the extent, as well as mechanistic and structural bases, of their substrate promiscuity. We expressed and purified 11 enzymes from seven subgroups of the flavin/deazaflavin oxidoreductase (FDOR) superfamily (A1, A2, A3, B1, B2, B3, B4) from the model soil actinobacterium Mycobacterium smegmatis. These enzymes reduced compounds from six chemical classes, including fundamental monocycles such as a cyclohexenone, a dihydropyran, and pyrones, as well as more complex quinone, coumarin, and arylmethane compounds. Substrate range and reduction rates varied between the enzymes, with the A1, A3, and B1 groups exhibiting greatest promiscuity. Molecular docking studies suggested that structurally diverse compounds are accommodated in the large substrate-binding pocket of the most promiscuous FDOR through hydrophobic interactions with conserved aromatic residues and the isoalloxazine headgroup of F420H2. Liquid chromatography-mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS) analysis of derivatized reaction products showed reduction occurred through a common mechanism involving hydride transfer from F420H- to the electron-deficient alkene groups of substrates. Reduction occurs when the hydride donor (C5 of F420H-) is proximal to the acceptor (electrophilic alkene of the substrate). These findings suggest that engineered actinobacterial F420H2-dependent reductases are promising novel biocatalysts for the facile transformation of a wide range of α,ß-unsaturated compounds.

Molecular basis for the behavioral effects of the odorant degrading enzyme Esterase 6 in Drosophila.

Previous electrophysiological and behavioural studies implicate esterase 6 in the processing of the pheromone cis-vaccenyl acetate and various food odorants that affect aggregation and reproductive behaviours. Here we show esterase 6 has relatively high activity against many of the short-mid chain food esters, but negligible activity against cis-vaccenyl acetate. The crystal structure of esterase 6 confirms its substrate-binding site can accommodate many short-mid chain food esters but not cis-vaccenyl acetate. Immunohistochemical assays show esterase 6 is expressed in non-neuronal cells in the third antennal segment that could be accessory or epidermal cells surrounding numerous olfactory sensilla, including basiconics involved in food odorant detection. Esterase 6 is also produced in trichoid sensilla, but not in the same cell types as the cis-vaccenyl acetate binding protein LUSH. Our data support a model in which esterase 6 acts as a direct odorant degrading enzyme for many bioactive food esters, but not cis-vaccenyl acetate.

Binary combinations of organophosphorus and synthetic pyrethroids are more potent acetylcholinesterase inhibitors than organophosphorus and carbamate mixtures: An in vitro assessment.

Anticholinesterase insecticides such as organophosphorous (OP) and carbamates pesticides (CB); and synthetic pyrethroids (SP) pesticides commonly co-occur in the environment. This raises the possibility of antagonistic, additive, or synergistic neurotoxicity in exposed organisms. Acetylcholinesterase (AChE) inhibition has been demonstrated to be useful as a biomarker for exposure to OP and CBs in many environments. This study investigated the response of housefly (Musca domestica) head AChE (HF-AChE) exposed to five OPs; chlorpyrifos (CPF), malathion (MLT), triazophos (TRZ), monocrotophos (MCP) and profenofos (PRF) and two CBs; carbaryl (CRB) and carbofuran (CBF) as individual compounds and as binary mixtures of OPs and CBs under in vitro conditions. In addition, the selected OPs and CBs were evaluated for their toxicity in binary combinations with two SPs; deltamethrin (DLT) and cypermethrin (CYP) at fixed concentrations of 0.1 and 10µg/L. The toxicological interaction of five OPs with two CBs pesticides was evaluated under oxidised and un-oxidised conditions using a toxic unit (TU) approach and a concentration addition (CA) model. Pyrethroid combinations were assessed only under oxidised conditions. Since OPs and CBs act by a similar mechanism of inhibition of AChE, a dose additive effect was expected, but not conclusively found. TRZ with either CBF or CRB exhibited synergism under oxidised and un-oxidised conditions but the degree of synergism was stronger under un-oxidised conditions. Additivity was exhibited by CBF+MCP, CRB+MCP, CRB+MLT and CBF+MCP under un-oxidised conditions and CRB+MCP and CRB+CPF under oxidised conditions. Pyrethorids in combination with OPs (TRZ, MLT and CPF) were highly synergistic. In the present study, we used pure housefly head AChE without any interference of monooxygenase and/or esterase enzyme activities. Therefore these other enzymes were not producing the observed deviations from concentration-addition in the binary combinations between OPs, CBs and SPs. The mechanisms of OP, CB and SP interactions in pesticide mixtures requires further investigation.