Biological Channeling of a Reactive Intermediate in the Bifunctional Enzyme DmpFG

It has been hypothesized that the bifunctional enzyme DmpFG channels its intermediate, acetaldehyde, from one active site to the next using a buried intermolecular channel identified in the crystal structure. This channel appears to switch between an open and a closed conformation depending on whether the coenzyme NAD(+) is present or absent. Here, we applied molecular dynamics and metadynamics to investigate channeling within DmpFG in both the presence and absence of NAD(+). We found that substrate channeling within this enzyme is energetically feasible in the presence of NAD(+) but was less likely in its absence. Tyr-291, a proposed control point at the channel's entry, does not appear to function as a molecular gate. Instead, it is thought to orientate the substrate 4-hydroxy-2-ketovalerate in DmpG before reaction occurs, and may function as a proton shuttle for the DmpG reaction. Three hydrophobic residues at the channel's exit appear to have an important role in controlling the entry of acetaldehyde into the DmpF active site

Biological Channeling of a Reactive Intermediate in the Bifunctional Enzyme DmpFG

AbstractIt has been hypothesized that the bifunctional enzyme DmpFG channels its intermediate, acetaldehyde, from one active site to the next using a buried intermolecular channel identified in the crystal structure. This channel appears to switch between an open and a closed conformation depending on whether the coenzyme NAD+ is present or absent. Here, we applied molecular dynamics and metadynamics to investigate channeling within DmpFG in both the presence and absence of NAD+. We found that substrate channeling within this enzyme is energetically feasible in the presence of NAD+ but was less likely in its absence. Tyr-291, a proposed control point at the channel's entry, does not appear to function as a molecular gate. Instead, it is thought to orientate the substrate 4-hydroxy-2-ketovalerate in DmpG before reaction occurs, and may function as a proton shuttle for the DmpG reaction. Three hydrophobic residues at the channel's exit appear to have an important role in controlling the entry of acetaldehyde into the DmpF active site

Structure-Function Relationships of the Neisserial EptA Enzyme Responsible for Phosphoethanolamine Decoration of Lipid A: Rationale for Drug Targeting

Bacteria cause disease by two general mechanisms: the action of their toxins on host cells and induction of a pro-inflammatory response that can lead to a deleterious cytokine/chemokine response (e.g., the so-called cytokine storm) often seen in bacteremia/septicemia. These major mechanisms may overlap due to the action of surface structures that can have direct and indirect actions on phagocytic or non-phagocytic cells. In this respect, the lipid A (endotoxin) component of lipopolysaccharide (LPS) possessed by Gram-negative bacteria has been the subject of literally thousands of studies over the past century that clearly identified it as a key virulence factor in endotoxic shock. In addition to its capacity to modulate inflammatory responses, endotoxin can also modulate bacterial susceptibility to host antimicrobials, such as the host defense peptides termed cationic antimicrobial peptides. This review concentrates on the phosphoethanolamine (PEA) decoration of lipid A in the pathogenic species of the genus Neisseria [N. gonorrhoeae and N. meningitidis]. PEA decoration of lipid A is mediated by the enzyme EptA (formerly termed LptA) and promotes resistance to innate defense systems, induces the pro-inflammatory response and can influence the in vivo fitness of bacteria during infection. These important biological properties have driven efforts dealing with the biochemistry and structural biology of EptA that will facilitate the development of potential inhibitors that block PEA addition to lipid A

PPARα and PPARγ activation is associated with pleural mesothelioma invasion but therapeutic inhibition is ineffective

Mesothelioma is a cancer that typically originates in the pleura of the lungs. It rapidly invades the surrounding tissues, causing pain and shortness of breath. We compared cell lines injected either subcutaneously or intrapleurally and found that only the latter resulted in invasive and rapid growth. Pleural tumors displayed a transcriptional signature consistent with increased activity of nuclear receptors PPARα and PPARγ and with an increased abundance of endogenous PPAR-activating ligands. We found that chemical probe GW6471 is a potent, dual PPARα/γ antagonist with anti-invasive and anti-proliferative activity in vitro. However, administration of GW6471 at doses that provided sustained plasma exposure levels sufficient for inhibition of PPARα/γ transcriptional activity did not result in significant anti-mesothelioma activity in mice. Lastly, we demonstrate that the in vitro anti-tumor effect of GW6471 is off-target. We conclude that dual PPARα/γ antagonism alone is not a viable treatment modality for mesothelioma

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

The crystal structure determination of cholesterol oxidase

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Structural and conformational energy studies of ligands of angiotensin converting enzyme and thermolysin

Bibliography: p. 122-126

Oxygen access to the active site of cholesterol oxidase through a narrow channel is gated by an Arg-Glu pair

Cholesterol oxidase is a monomeric flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. Two forms of the enzyme are known, one containing the cofactor non-covalently bound to the protein and one in which the cofactor is covalently linked to a histidine residue. The x-ray structure of the enzyme from Brevibacterium sterolicum containing covalently bound FAD has been determined and refined to 1.7-Å resolution. The active site consists of a cavity sealed off from the exterior of the protein. A model for the steroid substrate, cholesterol, can be positioned in the pocket revealing the structural factors that result in different substrate binding affinities between the two known forms of the enzyme. The structure suggests that Glu475, located at the active site cavity, may act as the base for both the oxidation and the isomerization steps of the catalytic reaction. A water-filled channel extending toward the flavin moiety, inside the substrate-binding cavity, may act as the entry point for molecular oxygen for the oxidative half-reaction. An arginine and a glutamate residue at the active site, found in two conformations are proposed to control oxygen access to the cavity from the channel. These concerted side chain movements provide an explanation for the biphasic mode of reaction with dioxygen and the ping-pong kinetic mechanism exhibited by the enzyme