Synthetic Riboswitches for the Analysis of tRNA Processing by eukaryotic RNase P Enzymes

Removal of the 5' leader region is an essential step in the maturation of tRNA molecules in all domains of life. This reaction is catalyzed by various RNase P activities, ranging from ribonucleoproteins with ribozyme activity to protein-only forms. In Escherichia coli, the efficiency of RNase P mediated cleavage can be controlled by computationally designed riboswitch elements in a ligand-dependent way, where the 5' leader sequence of a tRNA precursor is either sequestered in a hairpin structure or presented as a single-stranded region accessible for maturation. In the presented work, the regulatory potential of such artificial constructs is tested on different forms of eukaryotic RNase P enzymes – two protein-only RNase P enzymes (PRORP1 and PRORP2) from Arabidopsis thaliana and the ribonucleoprotein of Homo sapiens. The PRORP enzymes were analyzed in vitro as well as in vivo in a bacterial RNase P complementation system. We also tested in HEK293T cells whether the riboswitches remain functional with human nuclear RNase P. While the regulatory principle of the synthetic riboswitches applies for all tested RNase P enzymes, the results also show differences in the substrate requirements of the individual enzyme versions. Hence, such designed RNase P riboswitches represent a novel tool to investigate the impact of the structural composition of the 5'-leader on substrate recognition by different types of RNase P enzymes.

Antioxidant Defense during Recovery of Resurrection Plant Haberlea rhodopensis from Drought- and Freezing-Induced Desiccation

In this study, the contribution of nonenzymatic (ascorbate, glutathione) and enzymatic antioxidants (superoxide dismutase, catalase, glutathione reductase, glutathione S-transferase) in the first hours of recovery of the resurrection plant Haberlea rhodopensis from drought- and freezing-induced desiccation was assessed. The initial stage of recovery after desiccation is critical for plants, but less investigated. To better understand the alterations in the activity of antioxidant enzymes, their isoenzyme patterns were determined. Our results showed that ascorbate content remained high during the first 9 h of rehydration of desiccated plants and declined when the leaves′ water content significantly increased. The glutathione content remained high at the first hour of rehydration and then strongly decreased. The changes in ascorbate and glutathione content during recovery from drought- and freezing-induced desiccation showed great similarity. At the beginning of rehydration (1–5 h), the activities of antioxidant enzymes were significantly increased or remained as in dry plants. During 7–24 h of rehydration, certain differences in the enzymatic responses between the two plant groups were registered. The maintenance of a high antioxidant activity and upregulation of individual enzyme isoforms indicated their essential role in protecting plants from oxidative damage during the onset of recovery.

Experimental and computational investigation of enzyme functional annotations uncovers misannotation in the EC 1.1.3.15 enzyme class

Only a small fraction of genes deposited to databases have been experimentally characterised. The majority of proteins have their function assigned automatically, which can result in erroneous annotations. The reliability of current annotations in public databases is largely unknown; experimental attempts to validate the accuracy within individual enzyme classes are lacking. In this study we performed an overview of functional annotations to the BRENDA enzyme database. We first applied a high-throughput experimental platform to verify functional annotations to an enzyme class of S-2-hydroxyacid oxidases (EC 1.1.3.15). We chose 122 representative sequences of the class and screened them for their predicted function. Based on the experimental results, predicted domain architecture and similarity to previously characterised S-2-hydroxyacid oxidases, we inferred that at least 78% of sequences in the enzyme class are misannotated. We experimentally confirmed four alternative activities among the misannotated sequences and showed that misannotation in the enzyme class increased over time. Finally, we performed a computational analysis of annotations to all enzyme classes in the BRENDA database, and showed that nearly 18% of all sequences are annotated to an enzyme class while sharing no similarity or domain architecture to experimentally characterised representatives. We showed that even well-studied enzyme classes of industrial relevance are affected by the problem of functional misannotation.

The E. coli helicase does not use ATP during replication.

The replisome is responsible for replication of DNA in all domains of life, with several of its individual enzyme components relying on hydrolysis of nucleoside triphosphates to provide energy for replisome function. Half a century of biochemical studies have demonstrated a dependence on ATP as an energy source for helicases to unwind duplex DNA during replication. Through single-molecule visualization of DNA replication by the Escherichia coli replisome, we demonstrate that the DnaB helicase does not rely on hydrolysis of ATP (or any ribo-NTPs) in the context of the elongating replisome. We establish that nucleotide incorporation by the leading-strand polymerase is the main motor driving the replication process.

BRENDA, the ELIXIR core data resource in 2021: new developments and updates

The BRENDA enzyme database (https://www.brenda-enzymes.org), established in 1987, has evolved into the main collection of functional enzyme and metabolism data. In 2018, BRENDA was selected as an ELIXIR Core Data Resource. BRENDA provides reliable data, continuous curation and updates of classified enzymes, and the integration of newly discovered enzymes. The main part contains >5 million data for ∼90 000 enzymes from ∼13 000 organisms, manually extracted from ∼157 000 primary literature references, combined with information of text and data mining, data integration, and prediction algorithms. Supplements comprise disease-related data, protein sequences, 3D structures, genome annotations, ligand information, taxonomic, bibliographic, and kinetic data. BRENDA offers an easy access to enzyme information from quick to advanced searches, text- and structured-based queries for enzyme-ligand interactions, word maps, and visualization of enzyme data. The BRENDA Pathway Maps are completely revised and updated for an enhanced interactive and intuitive usability. The new design of the Enzyme Summary Page provides an improved access to each individual enzyme. A new protein structure 3D viewer was integrated. The prediction of the intracellular localization of eukaryotic enzymes has been implemented. The new EnzymeDetector combines BRENDA enzyme annotations with protein and genome databases for the detection of eukaryotic and prokaryotic enzymes.

The Impact of Post-Analytical Tools on New York Screening for Krabbe Disease and Pompe Disease

New York uses a two-tier assay to screen newborns for Krabbe disease and Pompe disease. Individual enzyme activities are measured in the first-tier, and specimens from newborns with low activity are reflexed to second tier Sanger sequencing of the associated gene. Using only this two-tiered approach, the screen positive and false positive rates were high. In this study, we added an additional step that examines the activity of four additional lysosomal enzymes. Results for all enzymes are integrated using the multivariate pattern recognition software called Collaborative Laboratory Integrated Reports (CLIR) to assess the risk for disease. Results after one year of screening using the new algorithm are compared to the prior year of screening without consideration of the additional enzymes and use of CLIR. With CLIR the number of babies referred for Krabbe disease was reduced by almost 80% (from 48 to 10) and the number of babies referred for Pompe disease was reduced by almost 32% (22 to 15).

Overexpression of Medicago sativa glutamate-semialdehyde aminotransferase (GSA) gene in tobacco increased photosynthesis efficiency

To investigate the effect of increased glutamate-semialdehyde aminotransferase (GSA) on photosynthetic capacity and growth, tobacco (Nicoliana tabacum L. Xanti) plants with increased levels of glutamate-semialdehyde aminotransferase protein were produced. This was achieved using a cassette composed of a full-length Medicago sative cDNA under the control of the cauliflower mosaic virus 35S promoter. The results revealed distinct impacts of GSA activity on photosynthesis rate and growth in GSA over expression tobacco plants. In transgenic plants with increased GSA activity, an increase in soluble and insoluble sugars accumulation was evident. Total biomass, leaf area, plant height and internode 3-4 were increased in GSA sense plants, compared with equivalent wild-type tobacco plants. Moreover, transgenic tobacco plants with increased GSA activity exhibit higher levels of 5-aminolevulinic acid (ALA) accumulation and increased in content of chlorophyll and carotenoids pigments. Collectively, our data suggest that higher level of GSA activity gives an advantage to photosynthesis, growth in tobacco plants. This work also provides a case study that an individual enzyme in the biosynthesis of chlorophyll pathway may serve as a useful target for genetic engineering to improve photosynthesis and growth in plants.HighlightOverexpression of glutamate-semialdehyde aminotransferase (GSA) increase photosynthetic capacity, growth in tobacco.

Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase

Staphylococcus aureus is an important pathogen and biofilm former. Biofilms cause problems in clinics and food production and are highly recalcitrant to antibiotics and sanitizers. Bacteriophage endolysins kill bacteria by degrading their cell wall and are therefore deemed promising antimicrobials and anti-biofilm agents. Depolymerases targeting polysaccharides in the extracellular matrix have been suggested as parts of a multi-enzyme approach to eradicate biofilms. The efficacy of endolysins and depolymerases against S. aureus biofilms in static models has been demonstrated. However, there is a lack of studies evaluating their activity against biofilms grown under more realistic conditions. Here, we investigated the efficacy of the endolysin LysK and the poly-N-acetylglucosamine depolymerase DA7 against staphylococcal biofilms in static and dynamic (flow cell-based) models. LysK showed activity against multiple S. aureus strains, and both LysK and DA7 removed static and dynamic biofilms from polystyrene and glass surfaces at low micromolar and nanomolar concentrations, respectively. When combined, the enzymes acted synergistically, as demonstrated by crystal violet staining of static biofilms, significantly reducing viable cell counts compared to individual enzyme treatment in the dynamic model, and confocal laser scanning microscopy. Overall, our results suggest that LysK and DA7 are potent anti-biofilm agents, alone and in combination.

Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase

In this paper, we report an example of the engineered expression of tetrameric β-galactosidase (β-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric β-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10−4 per base). In addition, we studied the kinetics of the mistranslated β-gal at the single-molecule level.