Characterisation and engineering of a thermophilic RNA ligase from Palaeococcus pacificus.

RNA ligases are important enzymes in molecular biology and are highly useful for the manipulation and analysis of nucleic acids, including adapter ligation in next-generation sequencing of microRNAs. Thermophilic RNA ligases belonging to the RNA ligase 3 family are gaining attention for their use in molecular biology, for example a thermophilic RNA ligase from Methanobacterium thermoautotrophicum is commercially available for the adenylation of nucleic acids. Here we extensively characterise a newly identified RNA ligase from the thermophilic archaeon Palaeococcus pacificus (PpaRnl).

An enzyme-centric approach for constructing an amperometric l-malate biosensor with a long and programmable linear range.

l-Malate is a key flavor enhancer and acidulant in the food and beverage industry, particularly winemaking. Enzyme-based amperometric biosensors offer convenience for monitoring its concentration. However, only a small number of off-the-shelf malate-oxidizing enzymes have been used in previous devices.

Ceftazidime resistance in Pseudomonas aeruginosa is multigenic and complex.

Pseudomonas aeruginosa causes a wide range of severe infections. Ceftazidime, a cephalosporin, is a key antibiotic for treating infections but a significant proportion of isolates are ceftazidime-resistant. The aim of this research was to identify mutations that contribute to resistance, and to quantify the impacts of individual mutations and mutation combinations.

Development of a compartmentalised self-replication protocol for selection of superior blunt-end DNA ligases.

DNA ligases are widely used in molecular biology to generate recombinant DNA. However, having evolved for nick-sealing, they are inefficient at catalysing the blunt-ended ligations that are critical to many biotechnological applications, including next-generation sequencing. To facilitate engineering of superior blunt-ended DNA ligases, we have developed and validated a compartmentalised self-replication protocol that can select for the most effective ligases from a library of variants.

Dan Tawfik's Lessons for Protein Engineers about Enzymes Adapting to New Substrates.

Natural evolution has been creating new complex systems for billions of years. The process is spontaneous and requires neither intelligence nor moral purpose but is nevertheless difficult to understand. The late Dan Tawfik spent years studying enzymes as they adapted to recognize new substrates.

Complex Loop Dynamics Underpin Activity, Specificity, and Evolvability in the (βα) Barrel Enzymes of Histidine and Tryptophan Biosynthesis.

Enzymes are conformationally dynamic, and their dynamical properties play an important role in regulating their specificity and evolvability. In this context, substantial attention has been paid to the role of ligand-gated conformational changes in enzyme catalysis; however, such studies have focused on tremendously proficient enzymes such as triosephosphate isomerase and orotidine 5'-monophosphate decarboxylase, where the rapid (μs timescale) motion of a single loop dominates the transition between catalytically inactive and active conformations. In contrast, the (βα)-barrels of tryptophan and histidine biosynthesis, such as the specialist isomerase enzymes HisA and TrpF, and the bifunctional isomerase PriA, are decorated by multiple long loops that undergo conformational transitions on the ms (or slower) timescale.

Draft Genome Sequence of a Novel Species Isolated from the Marine Macroalga (Oyster Thief).

In the process of studying the relationship between marine macroalgae and their bacterial symbionts, we isolated a new species of , which we designated sp. nov. C1 (for " 1").

The hypothesized role of YbeZ in 16S rRNA maturation.

Ribosomes are the protein production machines in all living cells. Yet in contrast to our understanding of how the ribosome translates DNA information into life, the steps involved in ribosome biogenesis, the assembly of the ribosomal RNA (rRNA) and protein molecules that make up the ribosome, remain incomplete. YbeY is considered one of the most physiologically critical endoribonucleases and is implicated in numerous roles involving RNA including 16S rRNA maturation, yet our existing knowledge of its biochemical function fails to explain the phenotypes that manifest when it is lost.

Assessing the effectiveness of oxathiapiprolin toward , the causal agent of kauri dieback disease.

species cause disease and devastation of plants in ecological and horticultural settings worldwide. A recently identified species, , infects and ultimately kills the treasured kauri trees () that are endemic to New Zealand. Currently, there are few options for managing kauri dieback disease.

Complete Genome Sequence of a Novel Pseudomonas fluorescens Strain Isolated from the Flower of Kūmarahou (Pomaderris kumeraho).

Kūmarahou (Pomaderris kumeraho) is a shrub endemic to New Zealand used in rongoā (traditional medicine). While studying the antimicrobial properties of kūmarahou, we isolated a new strain of Pseudomonas fluorescens, which we designated KF1 (for "kūmarahou flower 1"). Here, we report the complete genome sequence of P.

Enzyme promiscuity in natural environments: alkaline phosphatase in the ocean.

Alkaline phosphatase (APase) is one of the marine enzymes used by oceanic microbes to obtain inorganic phosphorus (P) from dissolved organic phosphorus to overcome P-limitation. Marine APase is generally recognized to perform P-monoesterase activity. Here we integrated a biochemical characterization of a specific APase enzyme, examination of global ocean databases, and field measurements, to study the type and relevance of marine APase promiscuity.

Structures and kinetics of Thermotoga maritima MetY reveal new insights into the predominant sulfurylation enzyme of bacterial methionine biosynthesis.

Bacterial methionine biosynthesis can take place by either the trans-sulfurylation route or direct sulfurylation. The enzymes responsible for trans-sulfurylation have been characterized extensively because they occur in model organisms such as Escherichia coli. However, direct sulfurylation is actually the predominant route for methionine biosynthesis across the phylogenetic tree.

Enzyme-based amperometric biosensors for malic acid - A review.

Malic acid is a key flavour component of many fruits and vegetables. There is significant interest in technologies for monitoring its concentration, particularly in winemaking. In this review we systematically and comprehensively chart progress in the development of enzyme-based amperometric biosensors for malic acid.

Quantifying the taxonomic bias in enzymology.

The ongoing biotechnological revolution is rooted in our knowledge of enzymes. However, metagenomics is showing how little we know about Earth's enzyme repertoire. Deep sequencing has revolutionized our view of the tree of life.

Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active-site residues.

Selection for a promiscuous enzyme activity provides substantial opportunity for competition between endogenous and newly-encountered substrates to influence the evolutionary trajectory, an aspect that is often overlooked in laboratory directed evolution studies. We selected the nitro/quinone reductase NfsA for chloramphenicol detoxification by simultaneously randomising eight active-site residues and interrogating ~250,000,000 reconfigured variants. Analysis of every possible intermediate of the two best chloramphenicol reductases revealed complex epistatic interactions.

Methods for competitive enrichment and evaluation of superior DNA ligases.

DNA ligases have numerous applications in molecular biology and biotechnology. However, many of these applications require the ligation of blunt-ended DNA termini, which is an inefficient activity for existing commercial ligases. To address this limitation, we describe a compartmentalised self-replication protocol that enables enrichment of the most active ligase variants from an arrayed gene library, e.

Structure and kinetics of indole-3-glycerol phosphate synthase from : Decarboxylation is not essential for indole formation.

In tryptophan biosynthesis, the reaction catalyzed by the enzyme indole-3-glycerol phosphate synthase (IGPS) starts with a condensation step in which the substrate's carboxylated phenyl group makes a nucleophilic attack to form the pyrrole ring of the indole, followed by a decarboxylation that restores the aromaticity of the phenyl. IGPS from has the highest turnover number of all characterized IGPS enzymes, providing an excellent model system to test the necessity of the decarboxylation step. Since the 1960s, this step has been considered to be mechanistically essential based on studies of the IGPS-phosphoribosylanthranilate isomerase fusion protein from Here, we present the crystal structure of IGPS in complex with reduced CdRP, a nonreactive substrate analog, and using a sensitive discontinuous assay, we demonstrate weak promiscuous activity on the decarboxylated substrate 1-(phenylamino)-1-deoxyribulose-5-phosphate, with an ∼1000× lower rate of IGP formation than from the native substrate.

Distinct pseudokinase domain conformations underlie divergent activation mechanisms among vertebrate MLKL orthologues.

The MLKL pseudokinase is the terminal effector in the necroptosis cell death pathway. Phosphorylation by its upstream regulator, RIPK3, triggers MLKL's conversion from a dormant cytoplasmic protein into oligomers that translocate to, and permeabilize, the plasma membrane to kill cells. The precise mechanisms underlying these processes are incompletely understood, and were proposed to differ between mouse and human cells.

Assessment of Phenotype Microarray plates for rapid and high-throughput analysis of collateral sensitivity networks.

The crisis of antimicrobial resistance is driving research into the phenomenon of collateral sensitivity. Sometimes, when a bacterium evolves resistance to one antimicrobial, it becomes sensitive to others. In this study, we have investigated the utility of Phenotype Microarray (PM) plates for identifying collateral sensitivities with unprecedented throughput.

A large-scale whole-genome comparison shows that experimental evolution in response to antibiotics predicts changes in naturally evolved clinical .

is an opportunistic pathogen that causes a wide range of acute and chronic infections. An increasing number of isolates have mutations that make them antibiotic resistant, making treatment difficult. To identify resistance-associated mutations we experimentally evolved the antibiotic sensitive strain PAO1 to become resistant to three widely used anti-pseudomonal antibiotics, ciprofloxacin, meropenem and tobramycin.

Genomic and phenotypic comparison of environmental and patient-derived isolates of suggest that antimicrobial resistance is rare within the environment.

Patient-derived isolates of the opportunistic pathogen are frequently resistant to antibiotics due to the presence of sequence variants in resistance-associated genes. However, the frequency of antibiotic resistance and of resistance-associated sequence variants in environmental isolates of has not been well studied. Antimicrobial susceptibility testing (ciprofloxacin, ceftazidime, meropenem, tobramycin) of environmental (=50) and cystic fibrosis (=42) isolates was carried out.

The uncharacterized bacterial protein YejG has the same architecture as domain III of elongation factor G.

InterPro family IPR020489 comprises ~1000 uncharacterized bacterial proteins. Previously we showed that overexpressing the Escherichia coli representative of this family, EcYejG, conferred low-level resistance to aminoglycoside antibiotics. In an attempt to shed light on the biochemical function of EcYejG, we have solved its structure using multinuclear solution NMR spectroscopy.

Mechanisms of ciprofloxacin resistance in Pseudomonas aeruginosa: new approaches to an old problem.

The antibiotic ciprofloxacin is used extensively to treat a wide range of infections caused by the opportunistic pathogen Pseudomonas aeruginosa. Due to its extensive use, the proportion of ciprofloxacin-resistant P. aeruginosa isolates is rapidly increasing.

The BRD3 ET domain recognizes a short peptide motif through a mechanism that is conserved across chromatin remodelers and transcriptional regulators.

Members of the bromodomain and extra-terminal domain (BET) family of proteins (bromodomain-containing (BRD) 2, 3, 4, and T) are widely expressed and highly conserved regulators of gene expression in eukaryotes. These proteins have been intimately linked to human disease, and more than a dozen clinical trials are currently underway to test BET-protein inhibitors as modulators of cancer. However, although it is clear that these proteins use their bromodomains to bind both histones and transcription factors bearing acetylated lysine residues, the molecular mechanisms by which BET family proteins regulate gene expression are not well defined.