Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Accurate computation of thermodynamic activation parameters in the chorismate mutase reaction from empirical valence bond simulations

Chorismate mutase enzymes have long served as model systems for benchmarking new methods and tools in computational chemistry. Despite the enzymes’ prominence in literature, the extent of the roles activation enthalpy and entropy play in catalyzing the conversion of chorismite to prephenate is still subject to debate. Knowledge of these parameters is a key piece in fully understanding the mechanism of chorismite mutases. Within this study, we utilize EVB/MD free energy perturbations at a range of temperatures, allowing us to extract activation enthalpies and entropies from an Arrhenius plot of reaction free energies of the reaction catalyzed by a monofunctional Bacillus subtilis chorismate mutase and the promiscuous enzyme isochorismate pyruvate lyase of Pseudomonas aeruginosa. In comparison to the uncatalyzed reaction, our results show that both enzyme-catalyzed reactions exhibit a substantial reduction in activation enthalpy, while the effect on activation entropy is relatively minor, demonstrating that enzyme-catalyzed chorismate mutase reactions are enthalpically-driven. Furthermore, we observe that the monofunctional chorismate mutase from B. subtilis more efficiently catalyzes this reaction than its promiscuous counterpart. This is supported by a structural analysis of the reaction pathway at the transition state, from which we identified key residues explaining the enthalpically-driven nature of the reactions, and also the difference in efficiencies between the two enzymes

Computational design of the temperature optimum of an enzyme reaction

Cold-adapted enzymes are characterized both by a higher catalytic activity at low temperatures and by having their temperature optimum down-shifted, compared to mesophilic orthologs. In several cases, the optimum does not coincide with the onset of protein melting but reflects some other type of inactivation. In the psychrophilic a-amylase from an Antarctic bacterium, the inactivation is thought to originate from a specific enzyme-substrate interaction that breaks around room temperature. Here, we report a computational redesign of this enzyme aimed at shifting its temperature optimum upward. A set of mutations designed to stabilize the enzyme-substrate interaction were predicted by computer simulations of the catalytic reaction at different temperatures. The predictions were verified by kinetic experiments and crystal structures of the redesigned a-amylase, showing that the temperature optimum is indeed markedly shifted upward and that the critical surface loop controlling the temperature dependence approaches the target conformation observed in a mesophilic ortholog

Conformational tuning improves the stability of spirocyclic nitroxides with long paramagnetic relaxation times

Abstract Nitroxides are widely used as probes and polarization transfer agents in spectroscopy and imaging. These applications require high stability towards reducing biological environments, as well as beneficial relaxation properties. While the latter is provided by spirocyclic groups on the nitroxide scaffold, such systems are not in themselves robust under reducing conditions. In this work, we introduce a strategy for stability enhancement through conformational tuning, where incorporating additional substituents on the nitroxide ring effects a shift towards highly stable closed spirocyclic conformations, as indicated by X-ray crystallography and density functional theory (DFT) calculations. Closed spirocyclohexyl nitroxides exhibit dramatically improved stability towards reduction by ascorbate, while maintaining long relaxation times in electron paramagnetic resonance (EPR) spectroscopy. These findings have important implications for the future design of new nitroxide-based spin labels and imaging agents

Structure and Mechanism of a Cold-Adapted Bacterial Lipase

The structural origin of enzyme cold-adaptation has been the subject of considerable research efforts in recent years. Comparative studies of orthologous mesophilic-psychrophilic enzyme pairs found in nature are an obvious strategy for solving this problem, but they often suffer from relatively low sequence identity of the enzyme pairs. Small bacterial lipases adapted to distinctly different temperatures appear to provide an excellent model system for these types of studies, as they may show a very high degree of sequence conservation. Here, we report the first crystal structures of lipase A from the psychrophilic bacterium Bacillus pumilus, which confirm the high structural similarity to the mesophilic Bacillus subtilis enzyme, as indicated by their 81% sequence identity. We further employ extensive QM/MM calculations to delineate the catalytic reaction path and its energetics. The computational prediction of a rate-limiting deacylation step of the enzymatic ester hydrolysis reaction is verified by stopped-flow experiments, and steady-state kinetics confirms the psychrophilic nature of the B. pumilus enzyme. These results provide a useful benchmark for examining the structural basis of cold-adaptation and should now make it possible to disentangle the effects of the 34 mutations between the two enzymes on catalytic properties and thermal stability

Cryptic β-Lactamase Evolution Is Driven by Low β-Lactam Concentrations

Our current understanding of how low antibiotic concentrations shape the evolution of contemporary β-lactamases is limited. Using the widespread carbapenemase OXA-48, we tested the long-standing hypothesis that selective compartments with low antibiotic concentrations cause standing genetic diversity that could act as a gateway to developing clinical resistance. Here, we subjected Escherichia coli expressing blaOXA-48, on a clinical plasmid, to experimental evolution at sub-MICs of ceftazidime. We identified and characterized seven single variants of OXA-48. Susceptibility profiles and dose-response curves showed that they increased resistance only marginally. However, in competition experiments at sub-MICs of ceftazidime, they demonstrated strong selectable fitness benefits. Increased resistance was also reflected in elevated catalytic efficiencies toward ceftazidime. These changes are likely caused by enhanced flexibility of the Ω- and β5-β6 loops and fine-tuning of preexisting active site residues. In conclusion, low-level concentrations of β-lactams can drive the evolution of β-lactamases through cryptic phenotypes which may act as stepping-stones toward clinical resistance