Gating mechanism of elongating β-ketoacyl-ACP synthases.

Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesis. During de novo fatty acid and polyketide biosynthesis, β-ketoacyl-acyl carrier protein (ACP) synthases (KS), catalyze this process via a decarboxylative Claisen-like condensation reaction. KSs must recognize multiple chemically distinct ACPs and choreograph a ping-pong mechanism, often in an iterative fashion. Here, we report crystal structures of substrate mimetic bearing ACPs in complex with the elongating KSs from Escherichia coli, FabF and FabB, in order to better understand the stereochemical features governing substrate discrimination by KSs. Complemented by molecular dynamics (MD) simulations and mutagenesis studies, these structures reveal conformational states accessed during KS catalysis. These data taken together support a gating mechanism that regulates acyl-ACP binding and substrate delivery to the KS active site. Two active site loops undergo large conformational excursions during this dynamic gating mechanism and are likely evolutionarily conserved features in elongating KSs

Visualizing the Interface of Biotin and Fatty Acid Biosynthesis through SuFEx Probes

Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the Escherichia coli acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5′-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners. We further evaluate the BioF-AcpP interface through protein crystallography and mutational studies. Among the AcpP-interacting BioF surface residues, three critical arginine residues appear to be involved in AcpP recognition so that pimeloyl-AcpP can serve as the acyl donor for PLP-mediated catalysis. These findings validate an evolutionary gain-of-function for BioF, allowing the organism to build biotin directly from fatty acid biosynthesis through surface modifications selective for salt bridge formation with acidic AcpP residues.</p

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Accessing Nystatin through Mariculture

Understanding our oceans and their marine ecosystems has enabled the development of sustainable systems for mariculture. While the bulk of studies to date have focused on the production of food, its remarkable expanse has inspired the translation of other markets towards aquatic environments. This manuscript outlines an approach to pharmaceutical mariculture, by demonstrating a benchmark for future prototyping. Here, design, field evaluation and natural product chemistry are united to successfully produce nystatin at sea. This study begins by evaluating new designs for culture flasks, illustrating a next step towards developing self-contained bioreactors for culturing in marine environments. Through pilot studies, an underwater system was developed to cost effectively produce cultures that yielded 200 mg of nystatin per deployment. Overall, this study demonstrates the potential for the practical culturing of microbes in a marine environment and provides an important next step for the fledgling field of molecular mariculture

New Caledonian crows attend to multiple functional properties of complex tools

The ability to attend to the functional properties of foraging tools should affect energy-intake rates, fitness components and ultimately the evolutionary dynamics of tool-related behaviour. New Caledonian crows Corvus moneduloides use three distinct tool types for extractive foraging: non-hooked stick tools, hooked stick tools and tools cut from the barbed edges of Pandanus spp. leaves. The latter two types exhibit clear functional polarity, because of (respectively) a single terminal, crow-manufactured hook and natural barbs running along one edge of the leaf strip; in each case, the ‘hooks’ can only aid prey capture if the tool is oriented correctly by the crow during deployment. A previous experimental study of New Caledonian crows found that subjects paid little attention to the barbs of supplied (wide) pandanus tools, resulting in non-functional tool orientation during foraging. This result is puzzling, given the presumed fitness benefits of consistently orienting tools functionally in the wild. We investigated whether the lack of discrimination with respect to (wide) pandanus tool orientation also applies to hooked stick tools. We experimentally provided subjects with naturalistic replica tools in a range of orientations and found that all subjects used these tools correctly, regardless of how they had been presented. In a companion experiment, we explored the extent to which normally co-occurring tool features (terminal hook, curvature of the tool shaft and stripped bark at the hooked end) inform tool-orientation decisions, by forcing birds to deploy ‘unnatural’ tools, which exhibited these traits at opposite ends. Our subjects attended to at least two of the three tool features, although, as expected, the location of the hook was of paramount importance. We discuss these results in the context of earlier research and propose avenues for future work.Publisher PDFPeer reviewe