Sam Hay

Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation.

Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides. Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle. Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria, with substrates including polychlorinated biphenyls or dioxins. Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies. Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon–cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies, we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen–cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis.

Promoting motions in enzyme catalysis probed by pressure studies of kinetic isotope effects

Use of the pressure dependence of kinetic isotope effects, coupled with a study of their temperature dependence, as a probe for promoting motions in enzymatic hydrogen-tunneling reactions is reported. Employing morphinone reductase as our model system and by using stopped-flow methods, we measured the hydride transfer rate (a tunneling reaction) as a function of hydrostatic pressure and temperature. Increasing the pressure from 1 bar (1 bar = 100 kPa) to 2 kbar accelerates the hydride transfer reaction when both protium (from 50 to 161 s−1 at 25°C) and deuterium (12 to 31 s−1 at 25°C) are transferred. We found that the observed primary kinetic isotope effect increases with pressure (from 4.0 to 5.2 at 25°C), an observation incompatible with the Bell correction model for hydrogen tunneling but consistent with a full tunneling model. By numerical modeling, we show that both the pressure and temperature dependencies of the reaction rates are consistent with the framework of the environmentally coupled tunneling model of Kuznetsov and Ulstrup [Kuznetsov AM, Ulstrup J (1999) Can J Chem 77:1085–1096], providing additional support for the role of a promoting motion in the hydride tunneling reaction in morphinone reductase. Our study demonstrates the utility of “barrier engineering” by using hydrostatic pressure as a probe for tunneling regimes in enzyme systems and provides added and independent support for the requirement of promoting motions in such tunneling reactions.

New cofactor supports α,β-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition

The bacterial ubiD and ubiX or the homologous fungal fdc1 and pad1 genes have been implicated in the non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone (also known as coenzyme Q) biosynthesis or microbial biodegradation of aromatic compounds, respectively. Despite biochemical studies on individual gene products, the composition and cofactor requirement of the enzyme responsible for in vivo decarboxylase activity remained unclear. Here we show that Fdc1 is solely responsible for the reversible decarboxylase activity, and that it requires a new type of cofactor: a prenylated flavin synthesized by the associated UbiX/Pad1. Atomic resolution crystal structures reveal that two distinct isomers of the oxidized cofactor can be observed, an isoalloxazine N5-iminium adduct and a N5 secondary ketimine species with markedly altered ring structure, both having azomethine ylide character. Substrate binding positions the dipolarophile enoic acid group directly above the azomethine ylide group. The structure of a covalent inhibitor–cofactor adduct suggests that 1,3-dipolar cycloaddition chemistry supports reversible decarboxylation in these enzymes. Although 1,3-dipolar cycloaddition is commonly used in organic chemistry, we propose that this presents the first example, to our knowledge, of an enzymatic 1,3-dipolar cycloaddition reaction. Our model for Fdc1/UbiD catalysis offers new routes in alkene hydrocarbon production or aryl (de)carboxylation.

UbiX is a flavin prenyltransferase required for bacterial ubiquinone biosynthesis

Ubiquinone (also known as coenzyme Q) is a ubiquitous lipid-soluble redox cofactor that is an essential component of electron transfer chains. Eleven genes have been implicated in bacterial ubiquinone biosynthesis, including ubiX and ubiD, which are responsible for decarboxylation of the 3-octaprenyl-4-hydroxybenzoate precursor. Despite structural and biochemical characterization of UbiX as a flavin mononucleotide (FMN)-binding protein, no decarboxylase activity has been detected. Here we report that UbiX produces a novel flavin-derived cofactor required for the decarboxylase activity of UbiD. UbiX acts as a flavin prenyltransferase, linking a dimethylallyl moiety to the flavin N5 and C6 atoms. This adds a fourth non-aromatic ring to the flavin isoalloxazine group. In contrast to other prenyltransferases, UbiX is metal-independent and requires dimethylallyl-monophosphate as substrate. Kinetic crystallography reveals that the prenyltransferase mechanism of UbiX resembles that of the terpene synthases. The active site environment is dominated by π systems, which assist phosphate-C1′ bond breakage following FMN reduction, leading to formation of the N5–C1′ bond. UbiX then acts as a chaperone for adduct reorientation, via transient carbocation species, leading ultimately to formation of the dimethylallyl C3′–C6 bond. Our findings establish the mechanism for formation of a new flavin-derived cofactor, extending both flavin and terpenoid biochemical repertoires.

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.

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.

Are the catalytic properties of enzymes from piezophilic organisms pressure adapted

We report the crystal structure of dihydrofolate reductase (DHFR) from the psychropiezophilic bacterium Moritella profunda, which was isolated from the deep ocean at 2 °C and 280 bar. The structure is typical of a chromosomal DHFR and we were unable to identify any obvious structural features that would suggest pressure adaptation. In particular, the core regions of the enzyme are virtually identical to those of the DHFR from the mesophile Escherichia coli. The steady‐state rate at pH 9, which is limited by hydride transfer at atmospheric pressure, is roughly constant between 1 and 750 bar, falling at higher pressures. However, the value of KM increases with increasing pressure, and as a result kcat/KM decreases over the entire pressure range studied. Isotope effect studies showed that increasing the pressure causes a change in the rate‐limiting step of the reaction. We therefore see no evidence of pressure adaptation in either the structure or the activity of this enzyme.

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.

Inter-flavin electron transfer in cytochrome P450 reductase - effects of solvent and pH identify hidden complexity in mechanism.

This study on human cytochrome P450 reductase (CPR) presents a comprehensive analysis of the thermodynamic and kinetic effects of pH and solvent on two‐ and four‐electron reduction in this diflavin enzyme. pH‐dependent redox potentiometry revealed that the thermodynamic equilibrium between various two‐electron reduced enzyme species (FMNH•,FADH•; FMN,FADH2; FMNH2,FAD) is independent of pH. No shift from the blue, neutral di‐semiquinone (FMNH•,FADH•) towards the red, anionic species is observed upon increasing the pH from 6.5 to 8.5. Spectrophotometric analysis of events following the mixing of oxidized CPR and NADPH (1 to 1) in a stopped‐flow instrument demonstrates that the establishment of this thermodynamic equilibrium becomes a very slow process at elevated pH, indicative of a pH‐gating mechanism. The final level of blue di‐semiquinone formation is found to be pH independent. Stopped‐flow experiments using excess NADPH over CPR provide evidence that both pH and solvent significantly influence the kinetic exposure of the blue di‐semiquinone intermediate, yet the observed rate constants are essentially pH independent. Thus, the kinetic pH‐gating mechanism under stoichiometric conditions is of no significant kinetic relevance for four‐electron reduction, but rather modulates the observed semiquinone absorbance at 600 nm in a pH‐dependent manner. The use of proton inventory experiments and primary kinetic isotope effects are described as kinetic tools to disentangle the intricate pH‐dependent kinetic mechanism in CPR. Our analysis of the pH and isotope dependence in human CPR reveals previously hidden complexity in the mechanism of electron transfer in this complex flavoprotein.

Incorporation of hydrostatic pressure into models of hydrogen tunneling highlights a role for pressure-modulated promoting vibrations

Hydrostatic pressure offers an alternative to temperature as an experimental probe of hydrogen-transfer reactions. H tunneling reactions have been shown to exhibit kinetic isotope effects (KIEs) that are sensitive to pressure, and environmentally coupled H tunneling reactions, those reactions in which H transfer is coupled to atomic fluctuations (a promoting vibration) along the reaction coordinate, often have quite temperature-dependent KIEs. We present here a theoretical treatment of the combined effect of temperature and pressure on environmentally coupled H tunneling reactions. We develop a generalized expression for the KIE, which can be used as a simple fitting function for combined experimental temperature- and pressure-dependent KIE data sets. With this expression, we are able to extract information about the pressure dependence of both the apparent tunneling distance and the frequency of the promoting vibration. The KIE expression is tested on two data sets {the reduction of chloranil by leuco crystal violet [Isaacs, N. S., Javaid, K., and Rannala, E. (1998) J. Chem. Soc., Perkin Trans. 2, 709-711] and the reduction of morphinone reductase by NADH [Hay, S., Sutcliffe, M. J., and Scrutton, N. S. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 507-512]} and suggests that hydrostatic pressure is a sensitive probe of nuclear quantum mechanical effects in H-transfer reactions.