BioLindlar Catalyst: Ene-Reductase-Promoted Selective Bioreduction of Cyanoalkynes to Give (Z)-Cyanoalkenes.

The direct synthesis of alkenes from alkynes usually requires the use of transition-metal catalysts. Unfortunately, efficient biocatalytic alternatives for this transformation have yet to be discovered. Herein, the selective bioreduction of electron-deficient alkynes to alkenes catalysed by ene-reductases (EREDs) is described. Alkynes bearing ketone, aldehyde, ester, and nitrile moieties have been effectively reduced with excellent conversions and stereoselectivities, observing clear trends for the E/Z ratios depending on the nature of the electron-withdrawing group. In the case of cyanoalkynes, (Z)-alkenes were obtained as the major product, and the reaction scope was expanded to a wide variety of aromatic substrates (up to >99% conversion, and Z/E stereoselectivities of up to >99/1). Other alkynes containing aldehyde, ketone, or ester functionalities also proved to be excellent substrates, and interestingly gave the corresponding (E)-alkenes. Preparative biotransformations were performed on a 0.4 mmol scale, producing the desired (Z)-cyanoalkenes with good to excellent isolated yields (63-97%). This novel reactivity has been rationalised through molecular docking by predicting the binding poses of key molecules in the ERED-pu-0006 active site.

Implications of conformational flexibility, lipid binding, and regulatory domains in cell-traversal protein CelTOS for apicomplexan migration.

Malaria and other apicomplexan-caused diseases affect millions of humans, agricultural animals, and pets. Cell traversal is a common feature used by multiple apicomplexan parasites to migrate through host cells and can be exploited to develop therapeutics against these deadly parasites. Here, we provide insights into the mechanism of the Cell-traversal protein for ookinetes and sporozoites (CelTOS), a conserved cell-traversal protein in apicomplexan parasites and malaria vaccine candidate. CelTOS has previously been shown to form pores in cell membranes to enable traversal of parasites through cells. We establish roles for the distinct protein regions of Plasmodium vivax CelTOS and examine the mechanism of pore formation. We further demonstrate that CelTOS dimer dissociation is required for pore formation, as disulfide bridging between monomers inhibits pore formation, and this inhibition is rescued by disulfide-bridge reduction. We also show that a helix-destabilizing amino acid, Pro127, allows CelTOS to undergo significant conformational changes to assemble into pores. The flexible C terminus of CelTOS is a negative regulator that limits pore formation. Finally, we highlight that lipid binding is a prerequisite for pore assembly as mutation of a phospholipids-binding site in CelTOS resulted in loss of lipid binding and abrogated pore formation. These findings identify critical regions in CelTOS and will aid in understanding the egress mechanism of malaria and other apicomplexan parasites as well as have implications for studying the function of other essential pore-forming proteins.

A function of profilin in force generation during malaria parasite motility that is independent of actin binding.

During transmission of malaria-causing parasites from mosquito to mammal, Plasmodium sporozoites migrate at high speed within the skin to access the bloodstream and infect the liver. This unusual gliding motility is based on retrograde flow of membrane proteins and highly dynamic actin filaments that provide short tracks for a myosin motor. Using laser tweezers and parasite mutants, we previously suggested that actin filaments form macromolecular complexes with plasma membrane-spanning adhesins to generate force during migration. Mutations in the actin-binding region of profilin, a near ubiquitous actin-binding protein, revealed that loss of actin binding also correlates with loss of force production and motility. Here, we show that different mutations in profilin, that do not affect actin binding in vitro, still generate lower force during Plasmodium sporozoite migration. Lower force generation inversely correlates with increased retrograde flow suggesting that, like in mammalian cells, the slow down of flow to generate force is the key underlying principle governing Plasmodium gliding motility.

Inter-subunit interactions drive divergent dynamics in mammalian and Plasmodium actin filaments.

Cell motility is essential for protozoan and metazoan organisms and typically relies on the dynamic turnover of actin filaments. In metazoans, monomeric actin polymerises into usually long and stable filaments, while some protozoans form only short and highly dynamic actin filaments. These different dynamics are partly due to the different sets of actin regulatory proteins and partly due to the sequence of actin itself. Here we probe the interactions of actin subunits within divergent actin filaments using a comparative dynamic molecular model and explore their functions using Plasmodium, the protozoan causing malaria, and mouse melanoma derived B16-F1 cells as model systems. Parasite actin tagged to a fluorescent protein (FP) did not incorporate into mammalian actin filaments, and rabbit actin-FP did not incorporate into parasite actin filaments. However, exchanging the most divergent region of actin subdomain 3 allowed such reciprocal incorporation. The exchange of a single amino acid residue in subdomain 2 (N41H) of Plasmodium actin markedly improved incorporation into mammalian filaments. In the parasite, modification of most subunit-subunit interaction sites was lethal, whereas changes in actin subdomains 1 and 4 reduced efficient parasite motility and hence mosquito organ penetration. The strong penetration defects could be rescued by overexpression of the actin filament regulator coronin. Through these comparative approaches we identified an essential and common contributor, subdomain 3, which drives the differential dynamic behaviour of two highly divergent eukaryotic actins in motile cells.

A unique profilin-actin interface is important for malaria parasite motility.

Profilin is an actin monomer binding protein that provides ATP-actin for incorporation into actin filaments. In contrast to higher eukaryotic cells with their large filamentous actin structures, apicomplexan parasites typically contain only short and highly dynamic microfilaments. In apicomplexans, profilin appears to be the main monomer-sequestering protein. Compared to classical profilins, apicomplexan profilins contain an additional arm-like ß-hairpin motif, which we show here to be critically involved in actin binding. Through comparative analysis using two profilin mutants, we reveal this motif to be implicated in gliding motility of Plasmodium berghei sporozoites, the rapidly migrating forms of a rodent malaria parasite transmitted by mosquitoes. Force measurements on migrating sporozoites and molecular dynamics simulations indicate that the interaction between actin and profilin fine-tunes gliding motility. Our data suggest that evolutionary pressure to achieve efficient high-speed gliding has resulted in a unique profilin-actin interface in these parasites.

Identification of bacterial protease domains that cleave human IgM.

Immunoglobulin-degrading proteases are secreted by pathogenic bacteria to weaken the host immune response, contributing to immune evasion mechanisms during an infection. Proteases specific to IgG and IgA immunoglobulin classes have previously been identified and characterized, and only a single report exists on a porcine specific IgM-degrading enzyme. It is unclear whether human pathogens also produce enzymes that can break down human IgM. Here, we have identified four novel IgM-degrading proteases from different genera of human-infecting bacterial pathogens. All four protease domains cleave human IgM at a conserved and unique site in the constant region of IgM. These human IgM proteases may be a useful biochemical tool for the study of early immune responses and have therapeutic potential in IgM-mediated disease.

Sequence-structure-function characterization of the emerging tetracycline destructase family of antibiotic resistance enzymes.

Tetracycline destructases (TDases) are flavin monooxygenases which can confer resistance to all generations of tetracycline antibiotics. The recent increase in the number and diversity of reported TDase sequences enables a deep investigation of the TDase sequence-structure-function landscape. Here, we evaluate the sequence determinants of TDase function through two complementary approaches: (1) constructing profile hidden Markov models to predict new TDases, and (2) using multiple sequence alignments to identify conserved positions important to protein function. Using the HMM-based approach we screened 50 high-scoring candidate sequences in Escherichia coli, leading to the discovery of 13 new TDases. The X-ray crystal structures of two new enzymes from Legionella species were determined, and the ability of anhydrotetracycline to inhibit their tetracycline-inactivating activity was confirmed. Using the MSA-based approach we identified 31 amino acid positions 100% conserved across all known TDase sequences. The roles of these positions were analyzed by alanine-scanning mutagenesis in two TDases, to study the impact on cell and in vitro activity, structure, and stability. These results expand the diversity of TDase sequences and provide valuable insights into the roles of important residues in TDases, and flavin monooxygenases more broadly.

A new species of Xestophrys Redtenbacher, 1891 (Orthoptera: Tettigoniidae; Conocephalinae; Copiphorini) from West Bengal, India.

A new species, Xestophrys bengalensis sp. nov., from the West Bengal state of India is described in this paper. The new species is superficially similar to the Indonesian species Xestophrys javanicus lombockensis Carl, 1908, but differs from the latter in the smaller size, anterior femur unarmed on the external margin, and hind femur with two spines on the internal margin. A key to the species of Xestophrys Redtenbacher, 1891 is also provided.

A new species of Euconocephalus Karny, 1907 (Orthoptera: Tettigoniidae: Conocephalinae: Copiphorini) from India.

A new species, Euconocephalus narayanpurensis Kumar & Chand sp. nov., from India is described in this paper. The new species is similar to the African species Euconocephalus lineatipes (Bolívar, 1890), but differs from the latter in the smaller size, more acute humeral sinus, the narrowly rounded apex of elytra and convex male last abdominal tergite. A key to the Indian species of Euconocephalus Karny, 1907 is also provided.

Structure of anhydrotetracycline-bound Tet(X6) reveals the mechanism for inhibition of type 1 tetracycline destructases.

Inactivation of tetracycline antibiotics by tetracycline destructases (TDases) remains a clinical and agricultural threat. TDases can be classified as type 1 Tet(X)-like TDases and type 2 soil-derived TDases. Type 1 TDases are widely identified in clinical pathogens. A combination therapy of tetracycline and a TDase inhibitor is much needed to rescue the clinical efficacy of tetracyclines. Anhydrotetracycline is a pan-TDase inhibitor that inhibits both type 1 and type 2 TDases. Here, we present structural, biochemical, and phenotypic evidence that anhydrotetracycline binds in a substrate-like orientation and competitively inhibits the type 1 TDase Tet(X6) to rescue tetracycline antibiotic activity as a sacrificial substrate. Anhydrotetracycline interacting residues of Tet(X6) are conserved within type 1 TDases, indicating a conserved binding mode and mechanism of inhibition. This mode of binding and inhibition is distinct from anhydrotetracycline's inhibition of type 2 TDases. This study forms the framework for development of next-generation therapies to counteract enzymatic tetracycline resistance.

Structure-Based Design of Bisubstrate Tetracycline Destructase Inhibitors That Block Flavin Redox Cycling.

Tetracyclines (TCs) are an important class of antibiotics threatened by an emerging new resistance mechanism─enzymatic inactivation. These TC-inactivating enzymes, also known as tetracycline destructases (TDases), inactivate all known TC antibiotics, including drugs of last resort. Combination therapies consisting of a TDase inhibitor and a TC antibiotic represent an attractive strategy for overcoming this type of antibiotic resistance. Here, we report the structure-based design, synthesis, and evaluation of bifunctional TDase inhibitors derived from anhydrotetracycline (aTC). By appending a nicotinamide isostere to the C9 position of the aTC D-ring, we generated bisubstrate TDase inhibitors. The bisubstrate inhibitors have extended interactions with TDases by spanning both the TC and presumed NADPH binding pockets. This simultaneously blocks TC binding and the reduction of FAD by NADPH while "locking" TDases in an unproductive FAD "out" conformation.

Rediscovery of Liara (Unalianus) heteracanthus (Redtenbacher, 1891) (Orthoptera: Tettigoniidae: Conocephalinae: Agraeciini) from India with notes on its distribution.

More than one hundred years after its description, a male specimen of Liara(Unalianus)heteracanthus(Redtenbacher, 1891) was collected at a new locality in the Mizoram State, India. Opportunity is taken to redescribe and illustrate this specimen.

First description of the female of Letana mursinga Ingrisch amp; Shishodia, 2000 (Orthoptera: Tettigoniidae; Phaneropterinae; Letanini) from India.

Female of Letana mursinga Ingrisch Shishodia, 2000 is described first time from Arunachal Pradesh, India along with a checklist of Indian species of Letana Walker, 1869. A map is also prepared for its distribution.

First record of Orthelimaea himalayana (Ingrisch, 1990) (Orthoptera: Tettigoniidae: Phaneropterinae: Elimaeini) from India with description of its female.

Orthelimaea himalayana (Ingrisch, 1990) is recorded for the first time from India and its female is also described first time from Central Himalaya. A key to Indian species of Orthelimaea Karny, 1926 is also provided.

A new species from India with a key to all known species of the genus Anaptygus Mistshenko, 1951 (Orthoptera: Acrididae).

A new species of the genus Anaptygus Mistshenko, 1951, Anaptygus shishodiai Kumar Chandra sp. nov., from Valley of Flowers National Park, India is described in this paper. The new species is similar to A. qinghaiensis Yin, 1984, but differs from latter by length of fastigial foveolae 5.5 times its width in male and 3.2 times in female; apex of elytra reaching posterior margin of third abdominal tergite in male and reaching posterior margin of first abdominal tergite in female. A key to all the known species of Anaptygus Mistshenko, 1951 is also provided.

DNA barcoding of selected short-horned grasshoppers (Orthoptera: Acrididae) from Indian Himalayan region.

In the context of Indian zoogeography, the DNA barcode data of short-horned grasshoppers (family Acrididae) are limited in global databases. Hence, the present study was aimed to collect selected Acridid species from the Indian Himalayan regions and generate DNA barcode data to enrich the global database. The estimated K2P genetic distances, Bayesian analysis (BA) topology and multiple species delimitation methods (ABGD, bPTP, and GMYC) clearly discriminate all the studied species. Based on high genetic distance (7.5%), multiple clades, and more than one molecular operational taxonomic unit, the present study elucidates the allopatric speciation and presence of possible cryptic diversity of Oxya japonica within India, China, and Russia. The present study suggests the collection of multiple specimens from different geographical locations and the generation of more DNA barcode data would facilitate the actual diversity of this insect group.

Tetracycline-inactivating enzymes from environmental, human commensal, and pathogenic bacteria cause broad-spectrum tetracycline resistance.

Tetracycline resistance by antibiotic inactivation was first identified in commensal organisms but has since been reported in environmental and pathogenic microbes. Here, we identify and characterize an expanded pool of tet(X)-like genes in environmental and human commensal metagenomes via inactivation by antibiotic selection of metagenomic libraries. These genes formed two distinct clades according to habitat of origin, and resistance phenotypes were similarly correlated. Each gene isolated from the human gut encodes resistance to all tetracyclines tested, including eravacycline and omadacycline. We report a biochemical and structural characterization of one enzyme, Tet(X7). Further, we identify Tet(X7) in a clinical Pseudomonas aeruginosa isolate and demonstrate its contribution to tetracycline resistance. Lastly, we show anhydrotetracycline and semi-synthetic analogues inhibit Tet(X7) to prevent enzymatic tetracycline degradation and increase tetracycline efficacy against strains expressing tet(X7). This work improves our understanding of resistance by tetracycline-inactivation and provides the foundation for an inhibition-based strategy for countering resistance.

A new species of Xestophrys Redtenbacher, 1891 (Orthoptera: Tettigoniidae; Conocephalinae; Copiphorini) from India.

In the present paper a new species, Xestophrys namtseringa Kumar Chandra sp. nov. is described and illustrated from Indian Himalaya. Key to all the known species of Xestophrys Redtenbacher, 1891 is also provided. [Zoobank URL: urn:lsid:zoobank.org:act:C18B59A4-30F8-4753-8739-6482D7F5A6B2].

Functional genetic evaluation of DNA house-cleaning enzymes in the malaria parasite: dUTPase and Ap4AH are essential in Plasmodium berghei but ITPase and NDH are dispensable.

BACKGROUND: Cellular metabolism generates reactive oxygen species. The oxidation and deamination of the deoxynucleoside triphosphate (dNTP) pool results in the formation of non-canonical, toxic dNTPs that can cause mutations, genome instability, and cell death. House-cleaning or sanitation enzymes that break down and detoxify non-canonical nucleotides play major protective roles in nucleotide metabolism and constitute key drug targets for cancer and various pathogens. We hypothesized that owing to their protective roles in nucleotide metabolism, these house-cleaning enzymes are key drug targets in the malaria parasite. METHODS: Using the rodent malaria parasite Plasmodium berghei we evaluate here, by gene targeting, a group of conserved proteins with a putative function in the detoxification of non-canonical nucleotides as potential antimalarial drug targets: they are inosine triphosphate pyrophosphatase (ITPase), deoxyuridine triphosphate pyrophosphatase (dUTPase) and two NuDiX hydroxylases, the diadenosine tetraphosphate (Ap4A) hydrolase and the nucleoside triphosphate hydrolase (NDH). RESULTS: While all four proteins are expressed constitutively across the intraerythrocytic developmental cycle, neither ITPase nor NDH are required for parasite viability. dutpase and ap4ah null mutants, on the other hand, are not viable suggesting an essential function for these proteins for the malaria parasite. CONCLUSIONS: Plasmodium dUTPase and Ap4A could be drug targets in the malaria parasite.