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). Furthermore, electrostatic interactions between the terminal carboxylate and amino groups of the substrate and Arg297 and Glu81 impose substantial "strain" energy on the orientation of the cyclic intermediate to control its trajectory. In addition the "strain" energy, which appears to be sensitive to both the number of carbon atoms in the substrate/analogue and the position of the radical intermediates, may play a key role in controlling the transition of the enzyme from the closed to the open state. Our results provide new insights into several aspects of the radical mechanism in aminomutase catalysis and broaden our understanding of cofactor PLP-dependent reactions.

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. Therefore, while we have previously demonstrated that pressure and temperature dependences can be used to infer H-tunneling and the involvement of promoting vibrations, these effects should not be used in the absence of atomistic insight, as they can vary greatly for different enzymes. We show that a pressure-dependent KIE is not a definitive hallmark of quantum mechanical H-tunneling during an enzyme-catalyzed reaction and that pressure-independent KIEs cannot be used to exclude tunneling contributions or a role for promoting vibrations in the enzyme-catalyzed reaction. We conclude that coupling of MD calculations with experimental rate and KIE studies is required to provide atomistic understanding of pressure effects in enzyme-catalyzed reactions.

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. This process is coupled to repositioning of the Ado moiety of AdoCbl from the eastern conformation to the northern conformation. Combined quantum mechanics and molecular mechanics calculations further indicate that in the open form, the protein environment does not impact significantly on the Co-C bond homolytic rupture, rendering it unusually stable, and thus catalytically inactive. Upon formation of the closed form, the Co-C bond is activated through the synergy of steric and electrostatic effects arising from tighter interactions with the surrounding enzyme. The more pronounced effect of the protein in the closed form gives rise to an elongated Co-C bond (by 0.03 Å), puckering of the ribose and increased "strain" energy on the Ado group and to a lesser extent the corrin ring. Our computational studies reveal novel strategies employed by AdoCbl-dependent enzymes in the control of radical catalysis.

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. This study investigates how electrostatic and hydrophobic interactions affect the conformation and function of the complex at physiological temperatures, using both experimental and computational methods. The results suggest that, first, different complex conformational states exist at equilibrium, and the major difference between these states is the degree of hydrophobic interactions. Second, the conformational change mimics the biological activity of the complex as measured by substrate binding at the same temperatures. Finally, molecular dynamics simulation and detailed energy decomposition analysis provided supporting evidence at the atomic level for the presence of an excited state of the complex, the formation of which is largely driven by the disruption of hydrophobic interactions. Taken together, this study indicates that the dynamics of the hydrophobic residues plays an important role in regulating the function of the Tim9-Tim10 complex.

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. gambiae but its role in detoxification is not clear. CYP6M2 was expressed in Escherichia coli and a structural model was produced to examine its role in pyrethroid metabolism. Both permethrin and deltamethrin were metabolized. Rates were enhanced by A. gambiae cytochrome b(5) with kinetic parameters of K(M)=11±1µM and k(cat)=6.1±0.4 per min for permethrin (1:1 cis-trans) and K(M)=2.0±0.3µM and k(cat)=1.2±0.1 per min for deltamethrin. Mass spectrometry and NMR analysis identified 4'-hydroxy deltamethrin and hydroxymethyl deltamethrin as major and minor deltamethrin metabolites respectively. Secondary breakdown products included cyano(3-hydroxyphenyl)methyl deltamethrate and deltamethric acid. CYP6M2 was most highly transcribed in the midgut and Malpighian tubules of adult A. gambiae, consistent with a role in detoxification. Our data indicates that CYP6M2 plays an important role in metabolic resistance to pyrethroids and thus an important target for the design of new tools to combat malaria.

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. Mutagenesis studies have suggested that MAL becomes active after phosphorylation of tyrosines and the computational studies presented here characterize the possible roles of two tyrosines-Tyr86 and Tyr106-in MAL activity. The effects of phosphorylation on the structure of MAL and on its binding with two binding partners MyD88 and SOCS1 are studied here. The results suggest that phosphorylation of Tyr86 leads to conformational changes in the BB loop of MAL, and this conformational switch forms the interface for binding with MyD88. Similarly, our results suggest that phosphorylation of Tyr106 contributes to the stability of MAL-MyD88 dimer formation, and may form a possible binding site for SOCS1. Thus, our study supports roles for tyrosines 86 and 106 in signaling pathways involving MAL, and hence as potential drug targets against hyperinflammatory response to infection by Plasmodium falciparum and vivax.

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. Using DFT vibrational analysis of a simple active site model, we posit that the decrease in α-2° KIE with pressure may arise due to a decrease in the vibrational coupling between the NADH primary (transferred) and secondary hydrogens in the 'tunnelling ready configuration', which more closely resembles the reactant state than the transition state.

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. By making targeted mutations in the active site of morphinone reductase we are able to alter the equilibrium of conformational states for the reactive complex in turn altering the donor-acceptor (D-A) distance for H-transfer. The sub-A changes which we induce are monitored using novel spectroscopic and kinetic "rulers". This new way of detecting variation in D-A distance allows us to analyze trends between D-A distance and the force constant of a compressive dynamical mode. We find that as the D-A distance decreases, the force constant for a compressive mode increases. Further, we demonstrate that-contrary to current dogma-compression may not always cause the magnitude of the primary kinetic isotope effect to decrease.

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. A particularly interesting type of complex is the metabolon, containing enzymes within the same metabolic pathway. There is reasonably good evidence that metabolons exist, but they have not been detected using high-thoughput assays, possibly because of their fragility.

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. We performed theoretical and computational studies on the effects of barrier compression on the shape of potential energy surfaces/reaction barriers for model (malonaldehyde and methane/methyl radical anion) and enzymatic (aromatic amine dehydrogenase) proton transfer systems. In all cases, we find that barrier compression is associated with an approximately linear decrease in the activation energy. For partially nonadiabatic proton transfers, we show that barrier compression enhances, to similar extents, the rate of classical and proton tunneling reactions. Our analysis suggests that barrier compression-through fast promoting vibrations, or other means-could be a general mechanism for enhancing the rate of not only tunneling, but also classical, proton transfers in enzyme catalysis.

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. Here we present a combined QM/MM-based approach that can be used to assign key resonances in the FTIR spectrum of tryptophan tryptophyl quinone (TTQ) in the TTQ-dependent quinoprotein aromatic amine dehydrogenase (AADH). We show that consideration of the cofactor alone is not sufficient to identify correctly the experimentally observed resonances-inclusion of the protein is required for this. However, to enable accurate peak assignment, a stepwise approach is needed that builds up increasing levels of complexity from a simple system. This study serves as a benchmark for future QM/MM-based studies to predict the spectroscopic changes during the interconversion of intermediates in the reductive half-reaction catalyzed by AADH, and more generally for using a combined QM/MM approach to calculate spectroscopic data of protein cofactors and cofactor-based adducts.

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. Thus far there has been limited evidence that promoting vibrations can increase the rate of H-transfer. In the present communication we examine the single hydride transfer from both NADPH and NADH to FMN in the reductive half-reaction of pentaerythritol tetranitrate reductase (PETNR). We find that there is a significant promoting vibration with NADPH but not with NADH and that the observed rate of hydride transfer is significantly (approximately 15x) faster with NADPH. We rule out differences in rate due to variation in driving force and the donor-acceptor distance, suggesting it is the promoting vibration with NADPH that is the origin of the increased observed rate. This study therefore provides direct evidence that promoting vibrations can lead to an increase in rate.

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). The calculations support earlier gas-phase density functional theory modeling and show that compound 0 is a sluggish oxidant that is unable to compete with compound I. Furthermore, compound I is shown to react with dimethyl sulfide via single-state reactivity on a dominant quartet spin state surface.

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.We show that hydride transfer from NADH to FMN catalysed by the N189A mutant of morphinone reductase occurs along parallel "chemical" pathways in a conformationally rich free-energy landscape. We have developed experimental kinetic and spectroscopic tools by using hydrostatic pressure to explore this free-energy landscape. The crystal structure of the N189A mutant enzyme in complex with the unreactive coenzyme analogue NADH(4) indicates that the nicotinamide moiety of the analogue is conformationally less restrained than the corresponding structure of the wild-type NADH(4) complex. This increased degree of conformational freedom in the N189A enzyme gives rise to the concept of multiple reactive configurations (MRCs), and we show that the relative population of these states across the free-energy landscape can be perturbed experimentally as a function of pressure. Specifically, the amplitudes of individual kinetic phases that were observed in stopped-flow studies of the hydride transfer reaction are sensitive to pressure; this indicates that pressure drives an altered distribution across the energy landscape. We show by absorbance spectroscopy that the loss of charge-transfer character of the enzyme-coenzyme complex is attributed to the altered population of MRCs on the landscape. The existence of a conformationally rich landscape in the N189A mutant is supported by molecular dynamics simulations at low and high pressure. The work provides firm experimental and computational support for the existence of parallel pathways arising from multiple conformational states of the enzyme-coenzyme complex. Hydrostatic pressure is a powerful and general probe of multidimensional energy landscapes that can be used to analyse experimentally parallel pathways for enzyme-catalysed reactions. We suggest that this is especially the case following directed mutation of a protein, which can lead to increased population of reactant states that are essentially inaccessible in the free-energy landscape of wild-type enzyme.

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. Inhibition of the activities of heterologously expressed CYP microsomes (CYPs 2D6, 3A4, 1A1, 1A2, 2C9, and 2C19) was monitored, and the most potent inhibition was found to be against CYP1A1, with IC (50) values of 9.2 microM and 11.9 microM for quassin and neoquassin, respectively. The moderate inhibition against the CYP1A1 isoform by quassin and neoquassin displayed partial competitive inhibition kinetics, with inhibition constants ( K(i)) of 10.8 +/- 1.6 microM, for quassin and competitive inhibition kinetics, with a K(i) of 11.3 +/- 0.9 microM, for neoquassin. We then docked these two inhibitors into the active site of a model of CYP1A1, which provided insight at the atomic level into the structure-activity relationship of quassinoids with respect to this important CYP isoform known to be an activator of carcinogens, thus providing a useful basis for the search for more potent inhibitors of CYP1A1 that may have implications in chemoprotection.

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. Here we report reactivity (driving force) studies of AADH with p-substituted phenylethylamines for which the kinetic isotope effect (approximately 16) accompanying C-H/C-(2)H bond breakage is elevated above the semi-classical limit. We show bond breakage occurs by quantum tunnelling and that within the context of the environmentally coupled framework for H-tunnelling the presence of the p-substituent places greater demand on the apparent need for fast promoting motions. The crystal structure of AADH soaked with phenylethylamine or methoxyphenylethylamine indicates that the structural change identified with p-substituted benzylamines should not limit the reaction with p-substituted phenylethylamines. This is consistent with the elevated kinetic isotope effects measured with p-substituted phenylethylamines. We find a good correlation in the rate constant for proton transfer with bond dissociation energy for the reactive C-H bond, consistent with a rate that is limited by a Marcus-like tunnelling mechanism. As the driving force becomes larger, the rate of proton transfer increases while the Marcus activation energy becomes smaller. This is the first experimental report of the driving force perturbation of H-tunnelling in enzymes using a series of related substrates. Our study provides further support for proton tunnelling in AADH.

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. Understanding and engineering such modulation by the protein environment of the flavin redox potential (DeltaE(o)) is valuable in biotechnology and device design. In this study we employed classical molecular dynamics free energy simulations (MDFES), within a thermodynamic integration (TI) formalism, to calculate the change in FMN first reduction potential (DeltaDeltaE(o)(ox/sq)) imparted by 6 flavoprotein active site mutations. The combined performance of the AMBER ff03 (protein) and GAFF (cofactor) force fields was benchmarked against experimental data for mutations close to the isoalloxazine re- and si-faces that perturb the wild-type DeltaE(o)(ox/sq) value in Anabaena flavodoxin. The classical alchemical approach used in this study overestimates the magnitude of DeltaE(o) values, in common with other studies. Nevertheless, chemically accurate DeltaDeltaE(o) values--calculated to within 1 kcal mol(-1) of the experimental value--were obtained for five of the six mutations studied. We have shown that this approach is practical for quantitative in silico screening of the effect of mutations on the first reduction potential where experimental values and structural data are available for the wild-type flavoprotein. This approach promises to be useful as an integral part of future interdisciplinary strategies to engineer desired thermodynamic properties in flavoproteins of biotechnological interest.