Convergence of Theory and Experiment on the Role of Preorganization, Quantum Tunneling, and Enzyme Motions into Flavoenzyme-Catalyzed Hydride Transfer

Hydride transfer is one of the most common reactions catalyzed by enzymatic systems, and it has become an object of study because of possible significant quantum tunneling effects. In the present work, we provide a combination of theoretical QM/MM simulations and experimental measurements of the rate constants and kinetic isotopic effects (KIEs) for the hydride transfer reaction catalyzed by morphinone reductase, MR. Quantum mechanical tunneling coefficients, computed in the framework of variational transition-state theory, play a significant role in this reaction, reaching values of 23.8 ± 5.5 for the lightest isotopologue—one of the largest values reported for enzymatic systems. This prediction is supported by the agreement between the theoretically predicted rate constants and the corresponding experimental values. Simulations indicate that the role of protein motions can be satisfactorily described as equilibrium fluctuations along the reaction coordinate, in line with a high degree of preorganization displayed by this enzyme.V.M. is grateful to the University of Bath for the award of a David Parkin Visiting Professorship. The authors thankfully acknowledge the computer resources, technical expertise and assistance provided by the “Centre de Calcul de la Universitat de València” through the use of Multivac and Tirant

The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care

PANC Study (Pancreatitis: A National Cohort Study): national cohort study examining the first 30 days from presentation of acute pancreatitis in the UK

Abstract Background Acute pancreatitis is a common, yet complex, emergency surgical presentation. Multiple guidelines exist and management can vary significantly. The aim of this first UK, multicentre, prospective cohort study was to assess the variation in management of acute pancreatitis to guide resource planning and optimize treatment. Methods All patients aged greater than or equal to 18 years presenting with acute pancreatitis, as per the Atlanta criteria, from March to April 2021 were eligible for inclusion and followed up for 30 days. Anonymized data were uploaded to a secure electronic database in line with local governance approvals. Results A total of 113 hospitals contributed data on 2580 patients, with an equal sex distribution and a mean age of 57 years. The aetiology was gallstones in 50.6 per cent, with idiopathic the next most common (22.4 per cent). In addition to the 7.6 per cent with a diagnosis of chronic pancreatitis, 20.1 per cent of patients had a previous episode of acute pancreatitis. One in 20 patients were classed as having severe pancreatitis, as per the Atlanta criteria. The overall mortality rate was 2.3 per cent at 30 days, but rose to one in three in the severe group. Predictors of death included male sex, increased age, and frailty; previous acute pancreatitis and gallstones as aetiologies were protective. Smoking status and body mass index did not affect death. Conclusion Most patients presenting with acute pancreatitis have a mild, self-limiting disease. Rates of patients with idiopathic pancreatitis are high. Recurrent attacks of pancreatitis are common, but are likely to have reduced risk of death on subsequent admissions. </jats:sec

Untangling heavy protein and cofactor isotope effects on enzyme catalyzed hydride transfer

“Heavy” (isotopically labeled) enzyme isotope effects offer a direct experimental probe of the role of protein vibrations on enzyme-catalyzed reactions. Here we have developed a strategy to generate isotopologues of the flavoenzyme pentaerythritol tetranitrate reductase (PETNR) where the protein and/or intrinsic flavin mononucleotide (FMN) cofactor are isotopically labeled with ²H, ¹⁵N, and ¹³C. Both the protein and cofactor contribute to the enzyme isotope effect on the reductive hydride transfer reaction, but their contributions are not additive and may partially cancel each other out. However, the isotope effect specifically arising from the FMN suggests that vibrations local to the active site play a role in the hydride transfer chemistry, while the protein-only “heavy enzyme” effect demonstrates that protein vibrations contribute to catalysis in PETNR. In all cases, enthalpy–entropy compensation plays a major role in minimizing the magnitude of “heavy enzyme” isotope effects. Fluorescence lifetime measurements of the intrinsic flavin mononucleotide show marked differences between “light” and “heavy” enzymes on the nanosecond–picosecond time scale, suggesting relevant time scale(s) for those vibrations implicated in the “heavy enzyme” isotope effect on the PETNR reaction

Histone H3 binding to the PHD1 domain of histone demethylase KDM5A enables active site remodeling.

Histone demethylase KDM5A removes methyl marks from lysine 4 of histone H3 and is often overexpressed in cancer. The in vitro demethylase activity of KDM5A is allosterically enhanced by binding of its product, unmodified H3 peptides, to its PHD1 reader domain. However, the molecular basis of this allosteric enhancement is unclear. Here we show that saturation of the PHD1 domain by the H3 N-terminal tail peptides stabilizes binding of the substrate to the catalytic domain and improves the catalytic efficiency of demethylation. When present in saturating concentrations, differently modified H3 N-terminal tail peptides have a similar effect on demethylation. However, they vary greatly in their affinity towards the PHD1 domain, suggesting that H3 modifications can tune KDM5A activity. Furthermore, hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) experiments reveal conformational changes in the allosterically enhanced state. Our findings may enable future development of anti-cancer therapies targeting regions involved in allosteric regulation

Viral E protein neutralizes BET protein-mediated post-entry antagonism of SARS-CoV-2.

Inhibitors of bromodomain and extraterminal domain (BET) proteins are possible anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here we show that BET proteins should not be inactivated therapeutically because they&nbsp;are critical antiviral factors at the post-entry level. Depletion of BRD3 or BRD4 in cells overexpressing ACE2 exacerbates SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon production&nbsp;induced by SARS-CoV-2, a process phenocopied by the envelope (E)&nbsp;protein previously identified as a possible "histone mimetic." E protein, in an acetylated form, directly binds the second bromodomain of&nbsp;BRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responses&nbsp;via BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment