Carboxylic acid reductase enzymes (CARs) meet the demand in synthetic chemistry for a green and regiospecific route to aldehydes from their respective carboxylic acids. However, relatively few of these enzymes have been characterized. A sequence alignment with members of the ANL (Acyl‐CoA synthetase/ NRPS adenylation domain/Luciferase) superfamily of enzymes shed light on CAR functional dynamics. Four unstudied enzymes were selected by using a phylogenetic analysis of known and hypothetical CARs, and for the first time, a thorough biochemical characterization was performed. Kinetic analysis of these enzymes with various substrates shows that they have a broad but similar substrate specificity. Electron‐rich acids are favored, which suggests that the first step in the proposed reaction mechanism, attack by the carboxylate on the α‐phosphate of adenosine triphosphate (ATP), is the step that determines the substrate specificity and reaction kinetics. The effects of pH and temperature provide a clear operational window for the use of these CARs, whereas an investigation of product inhibition by NADP+, adenosine monophosphate, and pyrophosphate indicates that the binding of substrates at the adenylation domain is ordered with ATP binding first. This study consolidates CARs as important and exciting enzymes in the toolbox for sustainable chemistry and provides specifications for their use as a biocatalyst.

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Myosin-5, kinesin-1 and myosin-17 cooperate in secretion of fungal chitin synthaseThe interactions between cytoskeletal motors are here explored in the context of polarized secretion in fungi, revealing unexpected complexity in the regulation of vesicle transport to and docking at the hyphal tip.

Impressive progress in genome sequencing, protein expression and high-throughput crystallography and NMR has radically transformed the opportunities to use protein three-dimensional structures to accelerate drug discovery, but the quantity and complexity of the data have ensured a central place for informatics. Structural biology and bioinformatics have assisted in lead optimization and target identification where they have well established roles; they can now contribute to lead discovery, exploiting high-throughput methods of structure determination that provide powerful approaches to screening of fragment binding.

The 22 members of the FGF family have been implicated in cell proliferation, differentiation, survival, and migration. They are required for both development and maintenance of vertebrates, demonstrating an exquisite pattern of affinities for both protein and proteoglycan receptors. FGF19, one of the most divergent human FGFs, is unique in binding solely to one receptor, FGFR4. We have used molecular replacement to solve the crystal structure of FGF19 at 1.3 Å resolution using five superimposed FGF structures as the search model. The structure shows that two novel disulfide bonds found in FGF19, one of which appears to be conserved among several of the other FGFs, stabilize extended loops. The key heparin-binding loops of FGF19 have radically different conformations and charge patterns, compared to other FGFs, correlating with the unusually low affinity of FGF19 for heparin. A model for the complex of FGF19 with FGFR4 demonstrates that unique sequences in both FGF19 and FGFR4 are key to the formation of the complex. The structure therefore offers a clear explanation for the unusual affinity of FGF19 for FGFR4 alone.