The logic of protein post-translational modifications (PTMs): Chemistry, mechanisms and evolution of protein regulation through covalent attachments.

Protein post-translational modifications (PTMs) play a crucial role in all cellular functions by regulating protein activity, interactions and half-life. Despite the enormous diversity of modifications, various PTM systems show parallels in their chemical and catalytic underpinnings. Here, focussing on modifications that involve the addition of new elements to amino-acid sidechains, I describe historical milestones and fundamental concepts that support the current understanding of PTMs. The historical survey covers selected key research programmes, including the study of protein phosphorylation as a regulatory switch, protein ubiquitylation as a degradation signal and histone modifications as a functional code. The contribution of crucial techniques for studying PTMs is also discussed. The central part of the essay explores shared chemical principles and catalytic strategies observed across diverse PTM systems, together with mechanisms of substrate selection, the reversibility of PTMs by erasers and the recognition of PTMs by reader domains. Similarities in the basic chemical mechanism are highlighted and their implications are discussed. The final part is dedicated to the evolutionary trajectories of PTM systems, beginning with their possible emergence in the context of rivalry in the prokaryotic world. Together, the essay provides a unified perspective on the diverse world of major protein modifications.

Self-Immolation of a Bacterial Dehydratase Enzyme by its Epoxide Product.

Disabling the bacterial capacity to cause infection is an innovative approach that has attracted significant attention to fight against superbugs. A relevant target for anti-virulence drug discovery is the type I dehydroquinase (DHQ1) enzyme. It was shown that the 2-hydroxyethylammonium derivative 3 has in vitro activity since it causes the covalent modification of the catalytic lysine residue of DHQ1. As this compound does not bear reactive electrophilic centers, how the chemical modification occurs is intriguing. We report here an integrated approach, which involves biochemical studies, X-ray crystallography and computational studies on the reaction path using combined quantum mechanics/molecular mechanics Umbrella Sampling Molecular Dynamics, that evidences that DHQ1 catalyzes its self-immolation by transforming the unreactive 2-hydroxyethylammonium group in 3 into an epoxide that triggers the lysine covalent modification. This finding might open opportunities for the design of lysine-targeted irreversible inhibitors bearing a 2-hydroxyethylammonium moiety as an epoxide proform, which to our knowledge has not been reported previously.

Activation of dioxygen by copper metalloproteins and insights from model complexes.

Nature uses dioxygen as a key oxidant in the transformation of biomolecules. Among the enzymes that are utilized for these reactions are copper-containing metalloenzymes, which are responsible for important biological functions such as the regulation of neurotransmitters, dioxygen transport, and cellular respiration. Enzymatic and model system studies work in tandem in order to gain an understanding of the fundamental reductive activation of dioxygen by copper complexes. This review covers the most recent advancements in the structures, spectroscopy, and reaction mechanisms for dioxygen-activating copper proteins and relevant synthetic models thereof. An emphasis has also been placed on cofactor biogenesis, a fundamentally important process whereby biomolecules are post-translationally modified by the pro-enzyme active site to generate cofactors which are essential for the catalytic enzymatic reaction. Significant questions remaining in copper-ion-mediated O2-activation in copper proteins are addressed.

Evidence for a boat conformation at the transition state of GH76 α-1,6-mannanases--key enzymes in bacterial and fungal mannoprotein metabolism.

α-Mannosidases and α-mannanases have attracted attention for the insight they provide into nucleophilic substitution at the hindered anomeric center of α-mannosides, and the potential of mannosidase inhibitors as cellular probes and therapeutic agents. We report the conformational itinerary of the family GH76 α-mannanases studied through structural analysis of the Michaelis complex and synthesis and evaluation of novel aza/imino sugar inhibitors. A Michaelis complex in an (O) S2 conformation, coupled with distortion of an azasugar in an inhibitor complex to a high energy B2,5 conformation are rationalized through ab initio QM/MM metadynamics that show how the enzyme surface restricts the conformational landscape of the substrate, rendering the B2,5 conformation the most energetically stable on-enzyme. We conclude that GH76 enzymes perform catalysis using an itinerary that passes through (O) S2 and B2,5 (≠) conformations, information that should inspire the development of new antifungal agents.

Direct evidence for a peroxide intermediate and a reactive enzyme-substrate-dioxygen configuration in a cofactor-free oxidase.

Cofactor-free oxidases and oxygenases promote and control the reactivity of O2 with limited chemical tools at their disposal. Their mechanism of action is not completely understood and structural information is not available for any of the reaction intermediates. Near-atomic resolution crystallography supported by in crystallo Raman spectroscopy and QM/MM calculations showed unambiguously that the archetypical cofactor-free uricase catalyzes uric acid degradation via a C5(S)-(hydro)peroxide intermediate. Low X-ray doses break specifically the intermediate C5-OO(H) bond at 100 K, thus releasing O2 in situ, which is trapped above the substrate radical. The dose-dependent rate of bond rupture followed by combined crystallographic and Raman analysis indicates that ionizing radiation kick-starts both peroxide decomposition and its regeneration. Peroxidation can be explained by a mechanism in which the substrate radical recombines with superoxide transiently produced in the active site.

Integrating genomics, molecular docking, and protein expression to explore new perspectives on polystyrene biodegradation.

Faced with the escalating challenge of global plastic pollution, this study specifically addresses the research gap in the biodegradation of polystyrene (PS). A PS-degrading bacterial strain was isolated from the gut of Tenebrio molitor, and genomics, molecular docking, and proteomics were employed to thoroughly investigate the biodegradation mechanisms of Pseudomonas putida H-01 against PS. Using scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (ATR-FTIR), and contact angle analysis, significant morphological and structural changes in the PS films under the influence of the H-01 strain were observed. The study revealed several potential degradation genes and ten enzymes that were specifically upregulated in the PS degradation environment. Additionally, a novel protein with laccase-like activity, LacQ1, was purified from this strain for the first time, and its crucial role in the PS degradation process was confirmed. Through molecular docking and molecular dynamics (MD) simulations, the interactions between the enzymes and PS were detailed, elucidating the binding and catalytic mechanisms of the degradative enzymes with the substrate. These findings have deepened our understanding of PS degradation.

Metalloprotein catalysis: structural and mechanistic insights into oxidoreductases from neutron protein crystallography.

Metalloproteins catalyze a range of reactions, with enhanced chemical functionality due to their metal cofactor. The reaction mechanisms of metalloproteins have been experimentally characterized by spectroscopy, macromolecular crystallography and cryo-electron microscopy. An important caveat in structural studies of metalloproteins remains the artefacts that can be introduced by radiation damage. Photoreduction, radiolysis and ionization deriving from the electromagnetic beam used to probe the structure complicate structural and mechanistic interpretation. Neutron protein diffraction remains the only structural probe that leaves protein samples devoid of radiation damage, even when data are collected at room temperature. Additionally, neutron protein crystallography provides information on the positions of light atoms such as hydrogen and deuterium, allowing the characterization of protonation states and hydrogen-bonding networks. Neutron protein crystallography has further been used in conjunction with experimental and computational techniques to gain insight into the structures and reaction mechanisms of several transition-state metal oxidoreductases with iron, copper and manganese cofactors. Here, the contribution of neutron protein crystallography towards elucidating the reaction mechanism of metalloproteins is reviewed.

Possible Effects of Dietary Anthocyanins on Diabetes and Insulin Resistance.

Diabetes is reaching epidemic proportions worldwide. Many dietary compounds have been found to exert health beneficial effects against different pathologies including diabetes. Most bioactive compounds have been identified in fruits and vegetables and their mechanisms of action explored both in vitro and in vivo. In particular, great interest has been given to polyphenols and especially to a specific subset of molecules, i.e. anthocyanins. Several lines of evidence suggest that anthocyanins have positive effects on human health by inducing a number of biological activities. This review will give an overview on the influence of dietary anthocyanins on preventing and managing type 2 diabetes. In particular, in vitro and in vivo studies will be presented. The article also reviews the potential clinical impact of the antidiabetic activity of anthocyanins and outlines the major challenges of using anthocyanins for diabetes treatment.

Cardioprotective efficacy of sevoflurane vs. propofol during induction and/or maintenance in patients undergoing coronary artery revascularization surgery without pump: A randomized trial.

PURPOSE: Pre and post-operative administration of sevoflurane in myocardial revascularization surgery provides enhanced cardioprotective effects exerted by pharmacologic pre- and post-conditioning, as compared to propofol. The identification of the enzymes involved in conditioning mechanisms is crucial to the understanding of the effects of sevoflurane in cardiac surgery patients. The impact of sevoflurane on another crucial target organ-the kidney-was also assessed. METHODS: Ninety patients undergoing off-pump myocardial revascularization surgery were allocated to receive either intra- and postoperative sevoflurane (SS), intraoperative sevoflurane and postoperative propofol (SP), or intra- and postoperative propofol (PP)). Troponin I and hemodynamic parameters were monitored during the first 48 postoperative hours; blood and urine samples were collected at baseline and at 24h to determine Akt, ERK1/2, PKG, iNO, bradykinin receptor, caspase 3, NT proBNP and urinary NGAL. RESULTS: The enzymes were overexpressed in the SS group, remained unchanged in the SP group, and decreased in the PP group. Renal function was best preserved in the SS group. CONCLUSIONS: The overexpression of enzymes induced by intraoperative anesthesia and postoperative sedation with sevoflurane reduces myocardial damage and improves renal function in patients undergoing off-pump myocardial revascularization surgery.

Unprecedented External Electric Field Effects on S-Nitrosothiols: Possible Mechanism of Biological Regulation?

Reactions of S-nitrosothiols (RSNOs), ubiquitous carriers of nitric oxide NO and its physiological activity, are tightly regulated in biological systems, but the mechanisms of this regulation are not well understood. Here, we computationally demonstrate that RSNO properties can be dramatically altered by biologically accessible external electric fields (EEFs) by modulation of the two minor antagonistic resonance structures of RSNOs, which have opposite formal charge distributions and bonding patterns. As these resonance contributions relate to the two competing modes of RSNO reactivity with nucleophiles, via N- or S-atom directed nucleophilic attack, EEFs are predicted to be efficient in controlling biologically important RSNO reactions with thiols. For instance, EEF catalysis might be one of the mechanisms behind the high selectivity of protein trans-S-nitrosation reactions, or putative nitroxyl HNO formation via RSNO S-thiolation reactions.