Quantum mechanics/molecular mechanics studies on the mechanism of action of cofactor pyridoxal 5'-phosphate in ornithine 4,5-aminomutase.

A computational study was performed on the experimentally elusive cyclisation step in the cofactor pyridoxal 5'-phosphate (PLP)-dependent D-ornithine 4,5-aminomutase (OAM)-catalysed reaction. Calculations using both model systems and a combined quantum mechanics/molecular mechanics approach suggest that regulation of the cyclic radical intermediate is achieved through the synergy of the intrinsic catalytic power of cofactor PLP and the active site of the enzyme. The captodative effect of PLP is balanced by an enzyme active site that controls the deprotonation of both the pyridine nitrogen atom (N1) and the Schiff-base nitrogen atom (N2).

Pressure effects on enzyme-catalyzed quantum tunneling events arise from protein-specific structural and dynamic changes.

The rate and kinetic isotope effect (KIE) on proton transfer during the aromatic amine dehydrogenase-catalyzed reaction with phenylethylamine shows complex pressure and temperature dependences. We are able to rationalize these effects within an environmentally coupled tunneling model based on constant pressure molecular dynamics (MD) simulations. As pressure appears to act anisotropically on the enzyme, perturbation of the reaction coordinate (donor-acceptor compression) is, in this case, marginal.

Large-scale domain conformational change is coupled to the activation of the Co-C bond in the B12-dependent enzyme ornithine 4,5-aminomutase: a computational study.

We present here an energetic and atomistic description of how D-ornithine 4,5-aminomutase (OAM), an adenosylcobalamin (AdoCbl; coenzyme B(12))-dependent isomerase, employs a large-scale protein domain conformational change to orchestrate the homolytic rupture of the Co-C bond. Our results suggest that in going from the open form (catalytically inactive) to the closed form (catalytically active), the Rossmann domain of OAM effectively approaches the active site as a rigid body. It undergoes a combination of a ~52° rotation and a ~14 Å translation to bring AdoCbl-initially positioned ~25 Å away-into the active-site cavity.

Temperature-dependent study reveals that dynamics of hydrophobic residues plays an important functional role in the mitochondrial Tim9-Tim10 complex.

Protein-protein interaction is a fundamental process in all major biological processes. The hexameric Tim9-Tim10 (translocase of inner membrane) complex of the mitochondrial intermembrane space plays an essential chaperone-like role during import of mitochondrial membrane proteins. However, little is known about the functional mechanism of the complex because the interaction is weak and transient.

Cytochrome P450 6M2 from the malaria vector Anopheles gambiae metabolizes pyrethroids: Sequential metabolism of deltamethrin revealed.

Resistance to pyrethroid insecticides in the malaria vector Anopheles gambiae is a major threat to malaria control programmes. Cytochome P450-mediated detoxification is an important resistance mechanism. CYP6M2 is over-expressed in wild populations of permethrin resistant A.

Probing active site geometry using high pressure and secondary isotope effects in an enzyme-catalysed 'deep' H-tunnelling reaction.

We report the first study of the effects of hydrostatic pressure on α-2° KIEs for an enzyme-catalysed H-transfer reaction that occurs by 'deep' tunnelling. High pressure causes a significant decrease in the observed α-2° KIE on the pre-steady-state hydride transfer from NADH to FMN in the flavoprotein morphinone reductase. We have recently shown that high pressure causes a reduction in macroscopic reaction barrier width for this reaction.

In-silico characterization of the effects of phosphorylated tyrosines 86 and 106 on structure and binding of MAL: insight into hyperinflammatory response to infection by the human malaria parasites.

The innate immune system uses inflammation to respond to infection of humans by various parasitic organisms and in some individuals can produce a hyperinflammatory response to infection by the human malaria parasites Plasmodium falciparum and vivax, leading to a more severe form of the disease-cerebral malaria (CM). Toll-like receptors (TLRs) 2 and 4 and members of its signaling pathway, including myeloid differentiation primary response protein (MyD88), MyD88 adapter-like protein (MAL) and suppressor of cytokine signaling 1 (SOCS1), are involved in this inflammatory response. A number of studies have suggested a possible role for MAL in developing CM and that modulating the behavior of MAL may prevent such complications.

Direct analysis of donor-acceptor distance and relationship to isotope effects and the force constant for barrier compression in enzymatic H-tunneling reactions.

The role of dynamical effects in enzyme catalysis is both complex and widely debated. Understanding how dynamics can influence the barrier to an enzyme catalyzed reaction requires the development of new methodologies and tools. In particular compressive dynamics-the focus of this study-may decrease both the height and width of a reaction barrier.

Protein-protein interactions.

In the present article, we describe the two standard high-throughput methods for identification of protein complexes: two-hybrid screens and TAP (tandem affinity purification) tagging. These methods have been used to characterize the interactome of Saccharomyces cerevisiae, showing that the majority of proteins are part of complexes, and that complexes typically consist of a core to which are bound 'party' and 'dater' proteins. Complexes typically are merely the sum of their parts.

New insights into the multi-step reaction pathway of the reductive half-reaction catalysed by aromatic amine dehydrogenase: a QM/MM study.

Computational insight into the multi-step reaction cycle of aromatic amine dehydrogenase is presented, identifying the energy landscape and pathway for multiple proton transfers. This atomistic picture of the reaction sequence--including short-lived reaction intermediates and a stepwise reaction mechanism--bridges the gap between a small number of crystallographic snapshots.

Barrier compression and its contribution to both classical and quantum mechanical aspects of enzyme catalysis.

It is generally accepted that enzymes catalyze reactions by lowering the apparent activation energy by transition state stabilization or through destabilization of ground states. A more controversial proposal is that enzymes can also accelerate reactions through barrier compression-an idea that has emerged from studies of H-tunneling reactions in enzyme systems. The effects of barrier compression on classical (over-the-barrier) reactions, and the partitioning between tunneling and classical reaction paths, have largely been ignored.

Assignment of the vibrational spectra of enzyme-bound tryptophan tryptophyl quinones using a combined QM/MM approach.

Fourier transform infrared (FTIR) spectroscopy can be used to provide a detailed time-resolved probe of reaction intermediates in enzyme-catalyzed systems. Accurate assignment of the respective chemical species being studied is key to the success of this approach. The plethora of signals from the protein environment, leading to complexity in the spectra, presents a particular challenge.

Evidence to support the hypothesis that promoting vibrations enhance the rate of an enzyme catalyzed H-tunneling reaction.

In recent years there has been a shift away from transition state theory models for H-transfer reactions. Models that incorporate tunneling as the mechanism of H-transfer are now recognized as a better description of such reactions. Central to many models of H-tunneling is the notion that specific vibrational modes of the protein and/or substrate can increase the probability of a H-tunneling reaction, modes that are termed promoting vibrations.

Quantum mechanics/molecular mechanics studies on the sulfoxidation of dimethyl sulfide by compound I and compound 0 of cytochrome P450: which is the better oxidant?

The cytochromes P450 are ubiquitous enzymes that are involved in key metabolizing processes in the body through the monoxygenation of substrates; however, their active oxidant is elusive. There have been reports that implicate that two oxidants, namely, the iron(IV)-oxo porphyrin cation radical (compound I) and the iron(III)-hydroperoxo complex (compound 0), both act as oxidants of sulfoxidation reactions, which contrasts theoretical studies on alkene epoxidation by compounds I and 0 that implicated compound 0 as a sluggish oxidant. To resolve this controversy and to establish the potency of compound I and compound 0 in sulfoxidation reactions, we have studied dimethyl sulfide sulfoxidation by both oxidants using the quantum mechanics/molecular mechanics (QM/MM) technique on cytochrome P450 enzymes and have set up a model of two P450 isozymes: P450(cam) and P450(BM3).

Parallel pathways and free-energy landscapes for enzymatic hydride transfer probed by hydrostatic pressure.

Mutation of an active-site residue in morphinone reductase leads to a conformationally rich landscape that enhances the rate of hydride transfer from NADH to FMN at standard pressure (1 bar). Increasing the pressure causes interconversion between different conformational substates in the mutant enzyme. While high pressure reduces the donor-acceptor distance in the wild-type enzyme, increased conformational freedom "dampens" its effect in the mutant.

Barrier compression enhances an enzymatic hydrogen-transfer reaction.

Putting the squeeze on: Hydrostatic pressure causes a shortening of the charge-transfer bond in the binary complex of morphinone reductase and NADH(4) (see diagram). Molecular dynamics simulations suggest that pressure reduces the average reaction barrier width by restricting the conformational space available to the flavin mononucleotide and NADH within the active site. The apparent rate of catalysis increases with pressure.

Inhibition of CYP1A1 by Quassinoids found in Picrasma excelsa.

Infusions of the plant Picrasma excelsa, known as Jamaican bitterwood tea, are commonly consumed to lower blood sugar levels in diabetics who are already on prescription medicines. We therefore investigated the inhibition properties of this tea against a panel of cytochrome P450 (CYP) enzymes, which are primarily responsible for the metabolism of a majority of drugs on the market. The two major ingredients, quassin and neoquassin, were then isolated and used for further characterization.

Driving force analysis of proton tunnelling across a reactivity series for an enzyme-substrate complex.

Quantitative structure-activity relationships are widely used to probe C-H bond breakage by quinoprotein enzymes. However, we showed recently that p-substituted benzylamines are poor reactivity probes for the quinoprotein aromatic amine dehydrogenase (AADH) because of a requirement for structural change in the enzyme-substrate complex prior to C-H bond breakage. This rearrangement is partially rate limiting, which leads to deflated kinetic isotope effects for p-substituted benzylamines.

Calculating chemically accurate redox potentials for engineered flavoproteins from classical molecular dynamics free energy simulations.

The tricyclic isoalloxazine nucleus of the redox cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) acts as an electron sink in life-sustaining biological electron transfer (eT). The functional diversity of flavin-containing proteins (flavoproteins) transcends that of free flavins. A large body of experimental evidence attributes natural control of flavoprotein-mediated eT to tuning of the thermodynamic driving force by the protein environment.

Solvent as a probe of active site motion and chemistry during the hydrogen tunnelling reaction in morphinone reductase.

The reductive half-reaction of morphinone reductase involves a hydride transfer from enzyme-bound beta-nicotinamide adenine dinucleotide (NADH) to a flavin mononucleotide (FMN). We have previously demonstrated that this step proceeds via a quantum mechanical tunnelling mechanism. Herein, we probe the effect of the solvent on the active site chemistry.

Multiple substrate binding by cytochrome P450 3A4: estimation of the number of bound substrate molecules.

Cytochrome P450 3A4, a major drug-metabolizing enzyme in man, is well known to show non-Michaelis-Menten steady-state kinetics for a number of substrates, indicating that more than one substrate can bind to the enzyme simultaneously, but it has proved difficult to obtain reliable estimates of exactly how many substrate molecules can bind. We have used a simple method involving studies of the effect of large inhibitors on the Hill coefficient to provide improved estimates of substrate stoichiometry from simple steady-state kinetics. Using a panel of eight inhibitors, we show that at least four molecules of the widely used CYP3A4 substrate 7-benzyloxyquinoline can bind simultaneously to the enzyme.

How do azoles inhibit cytochrome P450 enzymes? A density functional study.

To examine how azole inhibitors interact with the heme active site of the cytochrome P450 enzymes, we have performed a series of density functional theory studies on azole binding. These are the first density functional studies on azole interactions with a heme center and give fundamental insight into how azoles inhibit the catalytic function of P450 enzymes. Since azoles come in many varieties, we tested three typical azole motifs representing a broad range of azole and azole-type inhibitors: methylimidazolate, methyltriazolate, and pyridine.