Distinct Enzymes Research Articles

Plastidial (p)ppGpp Synthesis by the Ca2+-Dependent RelA-SpoT Homolog Regulates the Adaptation of Chloroplast Gene Expression to Darkness in Arabidopsis.

In bacteria, the hyper-phosphorylated nucleotide, guanosine 3',5'-bis(pyrophosphate) (ppGpp), functions as a secondary messenger under stringent conditions. ppGpp levels are controlled by two distinct enzymes, namely RelA and SpoT, in Escherichia coli. RelA-SpoT homologs (RSHs) are also conserved in plants where they function in the plastids. The model plant Arabidopsis thaliana contains four RSHs: RSH1, RSH2, RSH3 and Ca2+-dependent RSH (CRSH). Genetic characterizations of RSH1, RSH2 and RSH3 were undertaken, which showed that the ppGpp-dependent plastidial stringent response significantly influences plant growth and stress acclimation. However, the physiological significance of CRSH-dependent ppGpp synthesis remains unclear, as no crsh-null mutant has been available. Here, to investigate the function of CRSH, a crsh-knockout mutant of Arabidopsis was constructed using a site-specific gene-editing technique, and its phenotype was characterized. A transient increase in ppGpp was observed for 30 min in the wild type (WT) after the light-to-dark transition, but this increase was not observed in the crsh mutant. Similar analyses were performed with the rsh2-rsh3 double and rsh1-rsh2-rsh3 triple mutants and showed that the transient increments of ppGpp in the mutants were higher than those in the WT. The increase in ppGpp in the WT and rsh2 rsh3 accompanied decrements in the mRNA levels of some plastidial genes transcribed by the plastid-encoded plastid RNA polymerase. These results indicate that the transient increase in ppGpp at night is due to CRSH-dependent ppGpp synthesis and that the ppGpp level is maintained by the hydrolytic activities of RSH1, RSH2 and RSH3 to accustom plastidial gene expression to darkness.

Differential responses of genes and enzymes associated with ROS protective responses in the sugarcane smut fungus

The fungal pathogen Sporisorium scitamineum causes sugarcane smut disease. We have previously shown that resistant sugarcane plants induce ROS, coinciding with a delay in fungal colonization. Here, we investigated whether the fungus modifies the enzymatic antioxidant system in vitro and when colonizing sugarcane tissues in response to ROS. In vitro, the exposure to ROS did not affect cell integrity, and a combination of superoxide dismutases (SOD) and catalases (CAT) were active. In vitro, the fungus did not alter the expression of the transcriptional regulator Yap1 and the effector Pep1. The fungus activated distinct enzymes when colonizing plant tissues. Instead of CAT, S. scitamineum induced glutathione peroxidase (Gpx) expression only when colonizing smut-resistant plants. Yap1 had an earlier expression in both smut-susceptible and -resistant plants, with no apparent correlation with the expression of antioxidant genes sod, cat, gpx, or external redox imbalance. The expression of the effector pep1 was induced only in smut-resistant plants, potentially in response to ROS. These results collectively suggest that S. scitamineum copes with oxidative stress by inducing different mechanisms depending on the conditions (in vitro/in planta) and intensity of ROS. Moreover, the effector Pep1 is responsive to the stress imposed only by the sugarcane resistant genotype.

A Ten-Year Review of Carbapenemase Producing Enterobacterales (CPE) in London, United Kingdom

Background: To determine the pattern of CPE observed in a single region in the United Kingdom. Methods: From 2009 to 2018, clinical laboratories in England were requested to send suspected CPE from all sites to the national reference laboratory for confirmation and investigation of carbapenem resistance mechanism(s). Isolates of Enterobacterales from London laboratories and confirmed to have 1 or more carbapenemase genes were included in the analysis. Result: Between 2009 and 2018, 5,133 isolates were confirmed to produce a carbapenemase; at least 1 CPE was identified in every London Laboratory and hospital. Confirmations increased from 28 isolates in 2009 to 1857 in 2018 and with a sharp rise after the introduction of the ‘PHE toolkit’ in 2013 (Fig. 1). Most CPE (2655, 51.7%) were from rectal screens (the 3 most frequently identified carbapenemase families were OXA-48–like in 1,263 isolates, NDM in 971 and IMP in 128), 631 (12.3%) were from urine samples, 180 (3.5%) from blood cultures, 103 (2.0%) from sputum specimens and the remainder (1564, 30.5%) were swabs, fluids and tissues from various body sites. Moreover, 51 CPE (1%) were identified from environmental swabs. Isolates were predominantly Klebsiella spp (2,525, 49%; 2,088 were K. pneumoniae), followed by Escherichia coli (1,434, 27.9%), Enterobacter spp (746, 14.5%; 605 were E. cloacae complex), and Citrobacter spp (349, 6.8%); 10 other species contributed smaller numbers. Within the carbapenemase families, OXA-48-like enzymes predominated overall (2303, 44.9%), followed by NDM (1822, 35.5%), IMP (313, 6.1%), VIM (207, 4.0%), NDM+OXA-48-like (205, 4.0%), and KPC (196, 3.8%). The first detection of a CPE with 2 distinct enzymes occurred in 2012 (OXA-48-like and NDM) and since then 235 co-detections have been identified; 233 related to OXA-48-like with another gene. Conclusion: The first CPE isolate in London was identified in 2003, a Klebsiella spp with a VIM enzyme. The number of isolates submitted to the national reference laboratory has continued to increase year on year. VIM and NDM carbapenemases predominated in the early years, because of their association with several outbreaks; these have now been overwhelmed by OXA-48-like detections and outbreaks. The increasing numbers of CPE with a combination of a metallo- and a non-metallo carbapenemase increases the therapeutic challenges to treat infected patients. Bacteremia caused by CPE remains rare, suggesting that infection prevention and control efforts are having some impact. However, as colonization prevalence increases, the number of clinical infections will rise in the future unless control measures to limit transmission and spread are improved.Funding: NoneDisclosures: None

More than just a ticket canceller: the mitochondrial processing peptidase tailors complex precursor proteins at internal cleavage sites.

Most mitochondrial proteins are synthesized as precursors that carry N-terminal presequences. After they are imported into mitochondria, these targeting signals are cleaved off by the mitochondrial processing peptidase (MPP). Using the mitochondrial tandem protein Arg5,6 as a model substrate, we demonstrate that MPP has an additional role in preprotein maturation, beyond the removal of presequences. Arg5,6 is synthesized as a polyprotein precursor that is imported into mitochondria and subsequently separated into two distinct enzymes. This internal processing is performed by MPP, which cleaves the Arg5,6 precursor at its N-terminus and at an internal site. The peculiar organization of Arg5,6 is conserved across fungi and reflects the polycistronic arginine operon in prokaryotes. MPP cleavage sites are also present in other mitochondrial fusion proteins from fungi, plants, and animals. Hence, besides its role as a “ticket canceller” for removal of presequences, MPP exhibits a second conserved activity as an internal processing peptidase for complex mitochondrial precursor proteins.

Construction of Enzyme Nanoreactors to Enable Tumor Microenvironment Modulation and Enhanced Cancer Treatment.

Enzymes play pivotal roles in regulating and maintaining the normal functions of all living systems, and some of them are extensively employed for diagnosis and treatment of diverse diseases. More recently, several kinds of enzymes with unique catalytic activities have been found to be promising options to directly suppress tumor growth and/or augment the therapeutic efficacy of other treatments by modulating the hostile tumor microenvironment (TME), which is reported to negatively impair the therapeutic efficacy of different cancer treatments. In this review, first a summary is presented on the chemical approaches utilized for the construction of distinct enzyme nanoreactors with well-retained catalytic performance and reduced immunogenicity. Then, the utilization of such enzyme nanoreactors in attenuating tumor hypoxia, modulating extracellular matrix, and amplifying tumor oxidative stress is discussed in depth. Afterward, some perspectives are presented on the future development of such enzyme nanoreactors in TME modulation and enhanced cancer treatment.

Complex Evolution of Light-Dependent Protochlorophyllide Oxidoreductases in Aerobic Anoxygenic Phototrophs: Origin, Phylogeny, and Function.

Light-dependent protochlorophyllide oxidoreductase (LPOR) and dark-operative protochlorophyllide oxidoreductase are evolutionary and structurally distinct enzymes that are essential for the synthesis of (bacterio)chlorophyll, the primary pigment needed for both anoxygenic and oxygenic photosynthesis. In contrast to the long-held hypothesis that LPORs are only present in oxygenic phototrophs, we recently identified a functional LPOR in the aerobic anoxygenic phototrophic bacterium (AAPB) Dinoroseobacter shibae and attributed its presence to a single horizontal gene transfer event from cyanobacteria. Here, we provide evidence for the more widespread presence of genuine LPOR enzymes in AAPBs. An exhaustive bioinformatics search identified 36 putative LPORs outside of oxygenic phototrophic bacteria (cyanobacteria) with the majority being AAPBs. Using in vitro and in vivo assays, we show that the large majority of the tested AAPB enzymes are genuine LPORs. Solution structural analyses, performed for two of the AAPB LPORs, revealed a globally conserved structure when compared with a well-characterized cyanobacterial LPOR. Phylogenetic analyses suggest that LPORs were transferred not only from cyanobacteria but also subsequently between proteobacteria and from proteobacteria to Gemmatimonadetes. Our study thus provides another interesting example for the complex evolutionary processes that govern the evolution of bacteria, involving multiple horizontal gene transfer events that likely occurred at different time points and involved different donors.

Chain reactions: molecular mechanisms of RBR ubiquitin ligases

Ubiquitination is a fundamental post-translational modification that regulates almost all aspects of cellular signalling and is ultimately catalysed by the action of E3 ubiquitin ligases. The RING-between-RING (RBR) family of E3 ligases encompasses 14 distinct human enzymes that are defined by a unique domain organisation and catalytic mechanism. Detailed characterisation of several RBR ligase family members in the last decade has revealed common structural and mechanistic features. At the same time these studies have highlighted critical differences with respect to autoinhibition, activation and catalysis. Importantly, the majority of RBR E3 ligases remain poorly studied, and thus the extent of diversity within the family remains unknown. In this mini-review we outline the current understanding of the RBR E3 mechanism, structure and regulation with a particular focus on recent findings and developments that will shape the field in coming years.

Metatranscriptomics reveals the gene functions and metabolic properties of the major microbial community during Chinese Sichuan Paocai fermentation

Chinese Sichuan Paocai (CSP) is one of the world's best-known fermented vegetables with a large presence in the Chinese market. The dynamic microbial community is the main contributor to Paocai fermentation. However, little is known about the ecological distribution and functional importance of these community members. In this study, metatranscriptomics was used to comprehensively explore the active microbial community members and key transcripts with significant functions in the Paocai fermentation process. Enterobacter, Leuconostoc, and Lactobacillus dominated the three-fermentation stages (Pre-, Mid- and Lat-), respectively. Carbon metabolism was the most abundant pathway. GH (glycoside hydrolase) and GT (lycosyl transferase) were the two most highly expressed carbohydrate-active enzymes. The most highly differentially expressed genes were grouped in the biosynthesis of amino acids, followed by glycolysis. Meta-pathways in the Sichuan Paocai fermentation ecosystem were reconstructed, Lactobacillaceae and Enterobacteriaceae were the two most important metabolic contributors. In addition, the nrfA and nirB were two genes referred to distinct nitrite reductase enzymes and 9 specialized genes, such as eclo, ron and ent were expressed to produce autoinducer 2 (AI-2) kinase in response to population density. The present study revealed functional enzymes and meta-pathways of the active microbial communities, which provide a deeper understanding of their contribution to CSP products.

Comparative Studies of Vertebrate Mitochondrial Carbonic Anhydrase (CA5) Genes and Proteins: Evidence for Gene Duplication in Mammals with CA5A Being Liver Specific and CA5B Broadly Expressed and Located on the X-Chromosome

At least fifteen families of mammalian carbonic anhydrases (CA) (E.C. 4.2.1.2) catalyse the hydration of carbon dioxide and related functions. CA5A and CA5B genes encode distinct mitochondrial enzymes and perform essential biochemical roles, including ammonia detoxification and glucose metabolism. Bioinformatic methods were used to predict the amino acid sequences, secondary structures and gene locations for CA5A and CA5B genes and proteins using data from vertebrate genome projects. CA5A and CA5B genes usually contained 7 coding exons for each of the vertebrate genomes examined. Human CA5A and CA5B subunits contained 305 and 317 amino acids, respectively, with key amino acid residues including mitochondrial transit peptides; three Zinc binding sites (His130, His132, His155); and a Tyr164 active site. Phylogenetic analyses of vertebrate CA5 gene families suggested that it is an ancient gene in vertebrate evolution which had undergone a gene duplication event in a mammalian ancestral genome forming the CA5A and CA5B gene families in monotreme, marsupial and eutherian mammals. CA5A was predominantly expressed in liver whereas CA5B had a wide tissue distribution profile, was localized on the X-chromosome and was more highly conserved during mammalian evolution.

Yeast Intracellular Staining (yICS): Enabling High-Throughput, Quantitative Detection of Intracellular Proteins via Flow Cytometry for Pathway Engineering.

The complexities of pathway engineering necessitate screening libraries to discover phenotypes of interest. However, this approach is challenging when desirable phenotypes cannot be directly linked to growth advantages or fluorescence. In these cases, the ability to rapidly quantify intracellular proteins in the pathway of interest is critical to expedite the clonal selection process. While Saccharomyces cerevisiae remains a common host for pathway engineering, current approaches for intracellular protein detection in yeast either have low throughput, can interfere with protein function, or lack the ability to detect multiple proteins simultaneously. To fill this need, we developed yeast intracellular staining (yICS) that enables fluorescent antibodies to access intracellular compartments of yeast cells while maintaining their cellular integrity for analysis by flow cytometry. Using the housekeeping proteins β actin and glyceraldehyde 3-phophate dehydrogenase (GAPDH) as targets for yICS, we demonstrated for the first time successful antibody-based flow cytometric detection of yeast intracellular proteins with no modification. Further, yICS characterization of a recombinant d-xylose assimilation pathway showed 3-plexed, quantitative detection of the xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK) enzymes each fused with a small (6-10 amino acids) tag, revealing distinct enzyme expression profiles between plasmid-based and genome-integrated expression approaches. As a result of its high-throughput and quantitative capability, yICS enabled rapid screening of a library created from CRISPR-mediated XDH integration into the yeast δ site, identifying rare (1%) clones that led to an 8.4-fold increase in XDH activity. These results demonstrate the utility of yICS for greatly accelerating pathway engineering efforts, as well as any application where the high-throughput and quantitative detection of intracellular proteins is desired.

Continuous Flow Reactors from Microfluidic Compartmentalization of Enzymes within Inorganic Microparticles.

Compartmentalization and selective transport of molecular species are key aspects of chemical transformations inside the cell. In an artificial setting, the immobilization of a wide range of enzymes onto surfaces is commonly used for controlling their functionality but such approaches can restrict their efficacy and expose them to degrading environmental conditions, thus reducing their activity. Here, we employ an approach based on droplet microfluidics to generate enzyme-containing microparticles that feature an inorganic silica shell that forms a semipermeable barrier. We show that this porous shell permits selective diffusion of the substrate and product while protecting the enzymes from degradation by proteinases and maintaining their functionality over multiple reaction cycles. We illustrate the power of this approach by synthesizing microparticles that can be employed to detect glucose levels through simultaneous encapsulation of two distinct enzymes that form a controlled reaction cascade. These results demonstrate a robust, accessible, and modular approach for the formation of microparticles containing active but protected enzymes for molecular sensing applications and potential novel diagnostic platforms.

Characterization of different alginate lyases for dissolving Pseudomonas aeruginosa biofilms

Aggregates of Pseudomonas aeruginosa form a protective barrier against antibiotics and the immune system. These barriers, known as biofilms, are associated with several infectious diseases. One of the main components of these biofilms is alginate, a homo- and hetero-polysaccharide that consists of β-D-mannuronate (M) and α-L-guluronate (G) units. Alginate lyases degrade this sugar and have been proposed as biotherapeutic agents to dissolve P. aeruginosa biofilms. However, there are contradictory reports in the literature regarding the efficacy of alginate lyases against biofilms and their synergistic effect with antibiotics. We found that most positive reports used a commercial crude extract from Flavobacterium multivorum as the alginate lyase source. By using anion exchange chromatography coupled to nano LC MS/MS, we identified two distinct enzymes in this extract, one has both polyM and polyG (polyM/G) degradation activities and it is similar in sequence to a broad-spectrum alginate lyase from Flavobacterium sp. S20 (Alg2A). The other enzyme has only polyG activity and it is similar in sequence to AlyA1 from Zobellia galactanivorans. By characterizing both of these enzymes together with three recombinant alginate lyases (a polyM, a polyG and a polyM/G), we showed that only enzymes with polyM/G activity such as Alg2A and A1-II’ (alginate lyase from Sphingomonas sp.) are effective in dissolving biofilms. Furthermore, both activities are required to have a synergistic effect with antibiotics.

A new longitudinal variation in the structure of collagen fibrils and its relationship to locations of mechanical damage susceptibility

The hierarchical architecture of the collagen fibril is well understood, involving non-integer staggering of collagen molecules which results in a 67 nm periodic molecular density variation termed D-banding. Other than this variation, collagen fibrils are considered to be homogeneous at the micro-scale and beyond. Interestingly, serial kink structures have been shown to form at discrete locations along the length of collagen fibrils from some mechanically overloaded tendons. The formation of these kinks at discrete locations along the length of fibrils (discrete plasticity) may indicate pre-existing structural variations at a length scale greater than that of the D-banding. Using a high velocity nanomechanical mapping technique, 25 tendon collagen fibrils, were mechanically and structurally mapped along 10 μm of their length in dehydrated and hydrated states with resolutions of 20 nm and 8 nm respectively. Analysis of the variation in hydrated indentation modulus along individual collagen fibrils revealed a micro-scale structural variation not observed in the hydrated or dehydrated structural maps. The spacing distribution of this variation was similar to that observed for inter-kink distances seen in SEM images of discrete plasticity type damage. We propose that longitudinal variation in collagen fibril structure leads to localized mechanical susceptibility to damage under overload. Furthermore, we suggest that this variation has its origins in heterogeneous crosslink density along the length of collagen fibrils. The presence of pre-existing sites of mechanical vulnerability along the length of collagen fibrils may be important to biological remodeling of tendon, with mechanically-activated sites having distinct protein binding capabilities and enzyme susceptibility.

Targeting telomerase for its advent in cancer therapeutics.

Telomerase has emerged as an important primary target in anticancer therapy. It is a distinctive reverse transcriptase enzyme, which extends the length of telomere at the 3' chromosomal end, and uses telomerase reverse transcriptase (TERT) and telomerase RNA template-containing domains. Telomerase has a vital role and is a contributing factor in human health, mainly affecting cell aging and cell proliferation. Due to its unique feature, it ensures unrestricted cell proliferation in malignancy and plays a major role in cancer disease. The development of telomerase inhibitors with increased specificity and better pharmacokinetics is being considered to design and develop newer potent anticancer agents. Use of natural and synthetic compounds for the inhibition of telomerase activity can lead to an opening of new vistas in cancer treatment. This review details about the telomerase biochemistry, use of natural and synthetic compounds; vaccines and oncolytic virus in therapy that suppress the telomerase activity. We have discussed structure-activity relationships of various natural and synthetic telomerase inhibitors to help medicinal chemists and chemical biology researchers with a ready reference and updated status of their clinical trials. Suppression of human TERT (hTERT) activity through inhibition of hTERT promoter is an important approach for telomerase inhibition.

Laccase and catecholoxidase activities contribute to innate immunity in slipper limpets, Crepidula fornicata

The slipper limpet Crepidula fornicata is an invasive, non-native, marine species found throughout the coastal waters of southern England and Wales, UK. These limpets are considered to blight commercial shellfish banks, notably oysters, yet little is known about their disease-carrying capacity or their immunobiology. To address the latter, we isolated haemolymph (blood) from limpets and tested for the presence of the immune-enzyme phenoloxidase. Invertebrate phenoloxidases produce melanic polymers from simple phenolic substrates, which are deployed in the presence of pathogens because of their potent microbicidal and microbiostatic properties. We used a series of established substrates (e.g., tyrosine, hydroquinone) and inhibitors (e.g., 4-hexylresorcinol, benzoic acid) to target three distinct enzymes: laccase (para-diphenoloxidase), catecholoxidase (ortho-diphenoloxidase) and tyrosinase (monophenoloxidase). We confirmed laccase and catecholoxidase activities and characterised their kinetic properties across temperature and pH gradients (5–70 °C and 5–10, respectively). Crucially, we demonstrated that products derived from such laccase and catecholoxidase activities reduced significantly the numbers of colony-forming units of both Gram-positive and Gram-negative bacteria in vitro. We further screened limpet tissues for signs of melanin using wax histology, and found cells replete with eumelanin-like pigments and lipofuscin in the digestive gland, connective tissues, barrier epithelia and gills. Our data represent the first account of enzyme-based antibacterial defences, notably laccase, in C. fornicata.

The 18S ribosomal RNA m6 A methyltransferase Mettl5 is required for normal walking behavior in Drosophila.

RNA modifications have recently emerged as an important layer of gene regulation. N6-methyladenosine (m6 A) is the most prominent modification on eukaryotic messenger RNA and has also been found on noncoding RNA, including ribosomal and small nuclear RNA. Recently, several m6 A methyltransferases were identified, uncovering the specificity of m6 A deposition by structurally distinct enzymes. In order to discover additional m6 A enzymes, we performed an RNAi screen to deplete annotated orthologs of human methyltransferase-like proteins (METTLs) in Drosophila cells and identified CG9666, the ortholog of human METTL5. We show that CG9666 is required for specific deposition of m6 A on 18S ribosomal RNA via direct interaction with the Drosophila ortholog of human TRMT112, CG12975. Depletion of CG9666 yields a subsequent loss of the 18S rRNA m6 A modification, which lies in the vicinity of the ribosome decoding center; however, this does not compromise rRNA maturation. Instead, a loss of CG9666-mediated m6 A impacts fly behavior, providing an underlying molecular mechanism for the reported human phenotype in intellectual disability. Thus, our work expands the repertoire of m6 A methyltransferases, demonstrates the specialization of these enzymes, and further addresses the significance of ribosomal RNA modifications in gene expression and animal behavior.

PRMdb: A Repository of Predicted RNA Modifications in Plants.

Evidence is mounting that RNA modifications play essential roles in posttranscriptional regulation of gene expression. So far, over 150 RNA modifications catalyzed by distinct enzymes have been documented. In plants, genome-wide identification of RNA modifications is largely limited to the model species Arabidopsis thaliana, while lacking in diverse non-model plants. Here, we present PRMdb, a plant RNA modification database, based on the analysis of thousands of RNA-seq, degradome-seq and small RNA-seq data from a wide range of plant species using the well-documented tool HAMR (high-throughput analysis of modified ribonucleotide). PRMdb provides a user-friendly interface that enables easy browsing and searching of the tRNA and mRNA modification data. We show that PRMdb collects high-confidence RNA modifications including novel RNA modification sites that can be validated by genomic PCR and reverse transcription PCR. In summary, PRMdb provides a valuable web resource for deciphering the epitranscriptomes in diverse plant species and will facilitate functional studies of RNA modifications in plants. RPMdb is available via http://www.biosequencing.cn/PRMdb/.

An autoinhibitory mechanism in regulating m6A‐methylation of 28S rRNA

N6‐methyladenosine (m6A) modification represents an evolutionarily conserved epitranscriptomic mechanism that critically regulates RNA metabolism and function. m6A is widely present in mRNA and long noncoding RNA, as well as in ribosomal RNA (rRNA) and small nuclear RNA, mediated by an array of distinct writer enzymes. However, how m6A writers attain substrate specificities remains unclear. CCHC zinc finger‐containing protein ZCCHC4 is a newly identified RNA methyltransferase that specifically mediates the m6A modification on the site 4220 of 28S rRNA, which in turns impacts translation, cell proliferation and tumor growth. To provide mechanistic insights into the ZCCHC4‐mediated m6A RNA methylation, we performed structural characterization of the enzyme‐cofactor complex of human ZCCHC4. Our work reveals that the regulatory domains and the methyltransferase (MTase) domain pack into an integrated fold, thereby creating a multi‐domain RNA‐binding platform. Importantly, the MTase domain adopts an autoinhibitory conformation, in which the active site is occluded from RNA binding by an inter‐domain loop segment. Mutational and enzymatic analyses further indicate that this loop segment directly engages substrate recognition, providing an autoinhihitory mechanism by which the activity of ZCCHC4 is coupled with specific substrate binding. In addition, analysis of the substrate sequence and structure suggests a role of the stem‐loop structure within substrate in governing the substrate specificity. Together, our study uncovers an autoinhibitory mechanism that ensures specific interplay between ZCCHC4 and the RNA substrate, reminiscent of what was seen with the DNA methylation writers.Support or Funding InformationNIH 1R35GM119721

Characterization of the Tyrosyl‐S and Tyrosyl‐Z tRNA Synthetases from Pseudomonas aeruginosa and Discovery of Inhibitors

IntroductionPseudomonas aeruginosa, a Gram‐negative opportunistic pathogen, is a leading cause of nosocomial infections. The ability of this bacteria to form resilient biofilms on implanted medical devices results in high mortality rates. Patients with cystic fibrosis (CF) are especially at risk as P. aeruginosa colonizes the lungs of these patients forming biofilms which leads to chronic infections and is a leading cause of death. Aminoacyl‐tRNA synthetases (aaRSs) catalyze the covalent attachment of amino acids to their cognate tRNAs. These enzymes are essential for protein synthesis in bacteria and serve as validated targets for the development of new anti‐infectives. Unlike in other bacteria, the P. aeruginosa genome contains two genes (tyrS and tyrZ) which encode two distinct TyrRS enzymes, which are very different at the amino acid level containing only 27% conserved amino acid residues.Methods and ResultsThe kinetic parameters for the interaction of these enzymes with their three substrates (tRNA, ATP, tyrosine) were determined using timed aminoacylation and ATP:PPi exchange assays. The KM for interaction with tRNA, ATP, and tyrosine were 1.5, 204, 172 μM and 2.1, 496, 29 μM for TyrRS‐S and TyrRS‐Z, respectively. The kcat values for interaction with tRNA, ATP, tyrosine were 0.19, 1.0, 3.8 s−1 and 1.9, 3.8, 3.1 s−1 for TyrRS‐S and TyrRS‐Z, respectively. Both forms of TyrRS were developed into screening platforms using scintillation proximity assay (SPA) technology. Using this assay, a synthetic compound library from ChemDiv, Inc. containing over 2000 distinct compounds was screened to detect compounds with the ability to inhibit the function of TyrRS. Four compounds (BCD37H06, BCD38C11, BCD49D09, and BCD54B04) were confirmed as hit compounds. All four compounds inhibited the activity of TyrRS‐S and one of the compounds inhibited TyrRS‐Z. The compounds exhibited IC50 values in the low μM range. These compounds were found to have moderate broad spectrum inhibitory activity (MICs) against a panel of ten pathogenic bacteria. The compounds were all shown to be bacteriostatic in time‐kill kinetic assays. In competition assays, none of the compounds competed with tyrosine for binding, however BCD38C11 and BCE49D09 were competitive with ATP for binding. None of the compounds inhibited growth of human cell cultures.ConclusionTyrRS‐S and TyrRS‐Z from P. aeruginosa were characterized and used for development of screening platforms to identify compounds that have the potential for development as antibacterial agents against drug resistant pathogenic organisms.Support or Funding InformationThe authors are grateful for the financial support provided by the National Institutes of Health (grant number: 1SC3GM098173).

Ventromedial Hypothalamic Nucleus (VMN) Neuroestradiol: Sex Mono‐Versus Dimorphic Control of Glycogen Metabolism and Glucoregulatory Transmitter Signaling

The VMN functions within the neural glucostatic network. Estradiol exerts sex‐specific control of VMN nitric oxide (NO) and γ‐aminobutyric acid (GABA) transmission, which respectively stimulates or suppresses glucose counter‐regulatory hormone secretion. In the brain, the VMN is a principal site of neuroestradiol production by aromatase. This project utilized the aromatase inhibitor letrozole (LZ) alongside combinatory high‐resolution microdissection/Western blotting to address the premise that neuroestradiol governance of VMN metabolic sensory neuron protein marker [neuronal nitric oxide synthase (nNOS); glutamate decarboxylase65/67 (GAD)] expression is sex‐dimorphic. Glycogen metabolism impacts VMN nNOS and GAD profiles; here, LZ treatment effects on VMN glycogen synthase (GS) and phosphorylase brain‐ (GPBB; glucoprivic‐sensitive) and muscle (GPMM; norepinephrine‐sensitive) variant proteins were examined. VMN aromatase protein content was equivalent between the sexes; intracerebroventricular LZ infusion of testes‐intact male and ovariectomized, estradiol‐replaced female rats blocked insulin (INS)‐induced hypoglycemia up‐regulation of this profile. LZ elicited sex‐contingent changes in basal VMN nNOS and GAD expression, but blocked INS‐induced NO stimulation and GAD suppression in each sex. INS caused LZ‐reversible GPBB augmentation or suppression in male versus female, respectively. Male and female VMN GPMM content was correspondingly unchanged or decreased by INS; LZ reduced baseline GPMM expression in females only, but did not attenuate INS regulation in either sex. LZ correspondingly down‐ or up‐regulated baseline pyruvate recycling pathway marker protein expression in males (glutaminase) and females (malic enzyme‐1), and altered INS effects on the same proteins. Results show that neuroestradiol is required in each sex for optimal VMN metabolic transmitter signaling of hypoglycemia‐associated energy deficiency. Locally‐generated estradiol steroid involvement in sex‐specific hypoglycemic patterns of VMN glucoprivic‐sensitive GPBB expression may correlate with sex‐dimorphic glycogen mobilization during this metabolic stress. Neuroestradiol may also exert sex‐specific effects on glucogenic amino acid energy yield by actions on distinctive enzyme targets in each sex.Support or Funding InformationNational Institutes of Health DK‐109382