Cooperative Conformational Transitions Underpin the Activation Heat Capacity in the Temperature Dependence of Enzyme Catalysis.

Many enzymes display non-Arrhenius behavior with curved Arrhenius plots in the absence of denaturation. There has been significant debate about the origin of this behavior and recently the role of the activation heat capacity (ΔCP⧧) has been widely discussed. If enzyme-catalyzed reactions occur with appreciable negative values of ΔCP⧧ (arising from narrowing of the conformational space along the reaction coordinate), then curved Arrhenius plots are a consequence. To investigate these phenomena in detail, we have collected high precision temperature-rate data over a wide temperature interval for a model glycosidase enzyme MalL, and a series of mutants that change the temperature-dependence of the enzyme-catalyzed rate. We use these data to test a range of models including macromolecular rate theory (MMRT) and an equilibrium model. In addition, we have performed extensive molecular dynamics (MD) simulations to characterize the conformational landscape traversed by MalL in the enzyme-substrate complex and an enzyme-transition state complex. We have crystallized the enzyme in a transition state-like conformation in the absence of a ligand and determined an X-ray crystal structure at very high resolution (1.10 Å). We show (using simulation) that this enzyme-transition state conformation has a more restricted conformational landscape than the wildtype enzyme. We coin the term "transition state-like conformation (TLC)" to apply to this state of the enzyme. Together, these results imply a cooperative conformational transition between an enzyme-substrate conformation (ES) and a transition-state-like conformation (TLC) that precedes the chemical step. We present a two-state model as an extension of MMRT (MMRT-2S) that describes the data along with a convenient approximation with linear temperature dependence of the activation heat capacity (MMRT-1L) that can be used where fewer data points are available. Our model rationalizes disparate behavior seen for MalL and previous results for a thermophilic alcohol dehydrogenase and is consistent with a raft of data for other enzymes. Our model can be used to characterize the conformational changes required for enzyme catalysis and provides insights into the role of cooperative conformational changes in transition state stabilization that are accompanied by changes in heat capacity for the system along the reaction coordinate. TLCs are likely to be of wide importance in understanding the temperature dependence of enzyme activity and other aspects of enzyme catalysis.

Design and assembly of the 117-kb Phaeodactylum tricornutum chloroplast genome.

There is growing impetus to expand the repertoire of chassis available to synthetic biologists. Chloroplast genomes present an interesting alternative for engineering photosynthetic eukaryotes; however, development of the chloroplast as a synthetic biology chassis has been limited by a lack of efficient techniques for whole-genome cloning and engineering. Here, we demonstrate two approaches for cloning the 117-kb Phaeodactylum tricornutum chloroplast genome that have 90% to 100% efficiency when screening as few as 10 yeast (Saccharomyces cerevisiae) colonies following yeast assembly. The first method reconstitutes the genome from PCR-amplified fragments, whereas the second method involves precloning these fragments into individual plasmids from which they can later be released. In both cases, overlapping fragments of the chloroplast genome and a cloning vector are homologously recombined into a singular contig through yeast assembly. The cloned chloroplast genome can be stably maintained and propagated within Escherichia coli, which provides an exciting opportunity for engineering a delivery mechanism for bringing DNA directly to the algal chloroplast. Also, one of the cloned genomes was designed to contain a single SapI site within the yeast URA3 (coding for orotidine-5'-phosphate decarboxylase) open-reading frame, which can be used to linearize the genome and integrate designer cassettes via golden-gate cloning or further iterations of yeast assembly. The methods presented here could be extrapolated to other species-particularly those with a similar chloroplast genome size and architecture (e.g. Thalassiosira pseudonana).

Evidence for a short-lived resonance state in enzyme catalysis via rate-equation convolution.

At the cellular level, all biological function relies on enzymes to provide catalytic acceleration of essential biochemical processes driving cellular metabolism. The enzyme is presumed to lower the activation energy barrier separating reactants from products, but the precise mechanism remains unresolved. Here we examine the temperature dependence of the enzyme-catalyzed dissociation of p-nitrophenyl-α-D-glucopyranoside (pNPG), a chromogenic analog for maltose, isomaltose, and sucrose disaccharide sugars, into p-nitrophenol (pNP) and glucose (monosaccharide). The enzymes of interest are the wild type and mutant forms of glucosidase MalL produced by the probiotic bacterium Bacillus subtilis. The per-enzyme production rates k(T) for the pNPG→ glucose reaction all show a characteristic temperature profile with an Arrhenius-like (approximately exponential) slow acceleration at low temperatures, rising through a point of inflexion to reach a maximum, then turning over to decline steeply towards zero production at high temperatures. This asymmetric profile is found to be well fitted by convolving an exponential growth function f(T) with a Gaussian temperature distribution g(T) to produce an exponentially modified Gaussian function h(T). To give a physical interpretation of the convolution components, we make the temperature mapping Θ≡T_{ref}-T where T_{ref} marks the temperature at which a given mutant becomes fully denatured (unfolded) and therefore inactive, then convert the convolution components to probability density functions which obey the convolution theorem of statistics. Working in Θ space, we identify f(Θ) as the density function for an Arrhenius-like transition from ground-state A to metastable-state B, and g(Θ) as the Gaussian distribution of offset-temperature fluctuations for the metastable state. By mapping the standard thermodynamic relations for temperature and energy fluctuations to the enzyme frame of reference, we are able to derive an expression for the lifetime for the metastable B state. For the 15 enzyme experiments, we obtain a mean value 〈Δt〉≳(29.0±1.3)×10^{-15}s, in remarkably good agreement with the ∼30-fs estimate for the period of glycosidic bond oscillations extracted from published infrared spectroscopy. We suggest that the metastable B state provides a low-energy target that has the effect of lowering the activation energy barrier by presenting an alternative axis for the reaction coordinate.

Extinction dynamics: The interplay of species traits and the spatial scales of metapopulation declines.

Global changes can lead to species declines and extinctions through their impacts on species habitats at two distinct spatial scales: habitat destruction, in which individual habitat patches are destroyed by land-use change or natural disasters, and habitat degradation, in which larger scale changes, such as nitrogen deposition or climate change, lower mean population abundances across landscapes. We developed a theory showing that, even when these two forms of global change have an identical impact on a species' total amount of habitat, they have qualitatively different consequences for species dynamics and extinction. Using metapopulation theory and simulations, we found distinct impacts of these global changes characterized through several responses: the rate at which populations are lost from the remaining patches, extinction thresholds, and the duration of extinction debts. Habitat degradation causes a faster decline in species populations when habitat reduction is low, making it particularly detrimental for rare species. Habitat destruction has smaller impacts for low habitat reduction, but shows clear thresholds beyond which it surpasses degradation's negative impact; the location and steepness of the threshold depends on species dispersal, with poor dispersers having steeper thresholds. These results highlight the challenge of using population monitoring to assess the consequences of global changes and predict consequences of further change: extinction trajectories cannot be predicted due to thresholds (habitat destruction) and lagged dynamics that lead to extinction debts (habitat degradation). Our research clarifies why the impacts of one type of global change may poorly predict the impacts of the other and suggests general rules for predicting the long-term impacts of global changes based on species traits.

Combatting antimicrobial resistance via the cysteine biosynthesis pathway in bacterial pathogens.

Antibiotics are the cornerstone of modern medicine and agriculture, and rising antibiotic resistance is one the biggest threats to global health and food security. Identifying new and different druggable targets for the development of new antibiotics is absolutely crucial to overcome resistance. Adjuvant strategies that either enhance the activity of existing antibiotics or improve clearance by the host immune system provide another mechanism to combat antibiotic resistance. Targeting a combination of essential and non-essential enzymes that play key roles in bacterial metabolism is a promising strategy to develop new antimicrobials and adjuvants, respectively. The enzymatic synthesis of L-cysteine is one such strategy. Cysteine plays a key role in proteins and is crucial for the synthesis of many biomolecules important for defense against the host immune system. Cysteine synthesis is a two-step process, catalyzed by two enzymes. Serine acetyltransferase (CysE) catalyzes the first step to synthesize the pathway intermediate O-acetylserine, and O-acetylserine sulfhydrylase (CysK/CysM) catalyzes the second step using sulfide or thiosulfate to produce cysteine. Disruption of the cysteine biosynthesis pathway results in dysregulated sulfur metabolism, altering the redox state of the cell leading to decreased fitness, enhanced susceptibility to oxidative stress and increased sensitivity to antibiotics. In this review, we summarize the structure and mechanism of characterized CysE and CysK/CysM enzymes from a variety of bacterial pathogens, and the evidence that support targeting these enzymes for the development of new antimicrobials or antibiotic adjuvants. In addition, we explore and compare compounds identified thus far that target these enzymes.

Superior Conjugative Plasmids Delivered by Bacteria to Diverse Fungi.

Fungi are nature's recyclers, allowing for ecological nutrient cycling and, in turn, the continuation of life on Earth. Some fungi inhabit the human microbiome where they can provide health benefits, while others are opportunistic pathogens that can cause disease. Yeasts, members of the fungal kingdom, have been domesticated by humans for the production of beer, bread, and, recently, medicine and chemicals. Still, the great untapped potential exists within the diverse fungal kingdom. However, many yeasts are intractable, preventing their use in biotechnology or in the development of novel treatments for pathogenic fungi. Therefore, as a first step for the domestication of new fungi, an efficient DNA delivery method needs to be developed. Here, we report the creation of superior conjugative plasmids and demonstrate their transfer via conjugation from bacteria to 7 diverse yeast species including the emerging pathogen Candida auris. To create our superior plasmids, derivatives of the 57 kb conjugative plasmid pTA-Mob 2.0 were built using designed gene deletions and insertions, as well as some unintentional mutations. Specifically, a cluster mutation in the promoter of the conjugative gene traJ had the most significant effect on improving conjugation to yeasts. In addition, we created Golden Gate assembly-compatible plasmid derivatives that allow for the generation of custom plasmids to enable the rapid insertion of designer genetic cassettes. Finally, we demonstrated that designer conjugative plasmids harboring engineered restriction endonucleases can be used as a novel antifungal agent, with important applications for the development of next-generation antifungal therapeutics.

Arabidopsis PAP17 is a dual-localized purple acid phosphatase up-regulated during phosphate deprivation, senescence, and oxidative stress.

A 35 kDa monomeric purple acid phosphatase (APase) was purified from cell wall extracts of Pi starved (-Pi) Arabidopsis thaliana suspension cells and identified as AtPAP17 (At3g17790) by mass spectrometry and N-terminal microsequencing. AtPAP17 was de novo synthesized and dual-localized to the secretome and/or intracellular fraction of -Pi or salt-stressed plants, or senescing leaves. Transiently expressed AtPAP17-green fluorescent protein localized to lytic vacuoles of the Arabidopsis suspension cells. No significant biochemical or phenotypical changes associated with AtPAP17 loss of function were observed in an atpap17 mutant during Pi deprivation, leaf senescence, or salinity stress. Nevertheless, AtPAP17 is hypothesized to contribute to Pi metabolism owing to its marked up-regulation during Pi starvation and leaf senescence, broad APase substrate selectivity and pH activity profile, and rapid repression and turnover following Pi resupply to -Pi plants. While AtPAP17 also catalyzed the peroxidation of luminol, which was optimal at pH 9.2, it exhibited a low Vmax and affinity for hydrogen peroxide relative to horseradish peroxidase. These results, coupled with absence of a phenotype in the salt-stressed or -Pi atpap17 mutant, do not support proposals that the peroxidase activity of AtPAP17 contributes to the detoxification of reactive oxygen species during stresses that trigger AtPAP17 up-regulation.

Completeness and timeliness of diphtheria-tetanus-pertussis, measles-mumps-rubella, and polio vaccines in young children with chronic health conditions: A systematic review.

OBJECTIVE: To systematically review literature on uptake and timeliness of diphtheria-tetanus-pertussis, measles-mumps-rubella, and/or polio-containing vaccines ininfants who were born preterm, with a low birth weight, and/or with chronic health conditions that were diagnosed within the first 6 months of life. METHODS: Using a standardized search strategy developed by a medical librarian, records were extracted from MEDLINE, Embase, Database of Abstracts of Reviews of Effects, and CINAHL up to May 8, 2018. RESULTS: Out of the 1997 records that were screened, we identified 21 studies that met inclusion criteria. Eleven studies assessed vaccine coverage and/or timeliness in preterm infants, 6 in low birth weight infants, and 7 in children with chronic health conditions. Estimates of coverage in these populations were highly variable, ranging from 40% to 100% across the vaccines and population groups. CONCLUSIONS: There is a lack of studies reporting coverage and timeliness of routine immunizations in special populations of children. POLICY IMPLICATIONS: Our review suggests a need for improved surveillance of immunization status in special populations of infants, as wellas aneed for standardization of reporting practices.

Work-Life and Well-Being in U.K. Therapeutic Prison Officers: A Thematic Analysis.

Previous research has clearly demonstrated the positive impact of therapeutic interventions on offenders' well-being. Much less is known about the impact on prison staff facilitating and delivering such interventions. We employed qualitative methodology to capture a deeper understanding of the work of therapeutic prison officers. Seven prison officers working in a U.K. Category B therapeutic community prison were interviewed about their working lives, including their own participation in therapy. Following a thematic analysis approach, key findings indicated that the physical and cultural work environment was very important to staff; the therapeutic element of their job role, although demanding, was both satisfying and rewarding; and that working in a therapeutic prison environment provided the opportunity for personal as well as professional development. We conclude that further attention should be given to the unique nature of therapeutic prison work and the positive impact it can have on well-being at work.

Exposure of colonic epithelial cells to oxidative and endoplasmic reticulum stress causes rapid potassium efflux and calcium influx.

Endoplasmic reticulum (ER) stress and oxidative stress have recently been linked to the pathogenesis of inflammatory bowel diseases. Under physiological conditions, intestinal epithelial cells are exposed to ER and oxidative stress affecting the cellular ionic homeostasis. However, these altered ion flux 'signatures' during these stress conditions are poorly characterized. We investigated the kinetics of K(+) , Ca(2+) and H(+) ion fluxes during ER and oxidative stress in a colonic epithelial cell line LS174T using a non-invasive microelectrode ion flux estimation technique. ER and oxidative stress were induced by cell exposure to tunicamycin (TM) and copper ascorbate (CuAsc), respectively, from 1 to 24 h. Dramatic K(+) efflux was observed following acute ER stress with peak K(+) efflux being -30·6 and -138·7 nmolm(-2) s(-1) for 10 and 50 µg ml(-1) , respectively (p < 0·01). TM-dependent Ca(2+) uptake was more prolonged with peak values of 0·85 and 2·68 nmol m(-2) s(-1) for 10 and 50 µg ml(-1) TM, respectively (p < 0·02). Ion homeostasis was also affected by the duration of ER stress. Increased duration of TM treatment from 0 to 18 h led to increases in both K(+) efflux and Ca(2+) uptake. While K(+) changes were significantly higher at each time point tested, Ca(2+) uptake was significantly higher only after prolonged treatment (18 h). CuAsc also led to an increased K(+) efflux and Ca(2+) uptake. Functional assays to investigate the effect of inhibiting K(+) efflux with tetraethylammonium resulted in increased cell viability. We conclude that ER/oxidative stress in colonic epithelial cells cause dramatic K(+) , Ca(2+) and H(+) ion flux changes, which may predispose this lineage to poor stress recovery reminiscent of that seen in inflammatory bowel diseases.