Basile Khara

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.

Coupled Motions Direct Electrons along Human Microsomal P450 Chains

Directional electron transfer through biological redox chains can be achieved by coupling reaction chemistry to conformational changes in individual redox enzymes.

Ultrafast red light activation of Synechocystis phytochrome Cph1 triggers major structural change to form the Pfr signalling-competent state.

Phytochromes are dimeric photoreceptors that regulate a range of responses in plants and microorganisms through interconversion of red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Photoconversion between these states is initiated by light-driven isomerization of a bilin cofactor, which triggers protein structural change. The extent of this change, and how light-driven structural changes in the N-terminal photosensory region are transmitted to the C-terminal regulatory domain to initiate the signalling cascade, is unknown. We have used pulsed electron-electron double resonance (PELDOR) spectroscopy to identify multiple structural transitions in a phytochrome from Synechocystis sp. PCC6803 (Cph1) by measuring distances between nitroxide labels introduced into the protein. We show that monomers in the Cph1 dimer are aligned in a parallel ‘head-to-head’ arrangement and that photoconversion between the Pr and Pfr forms involves conformational change in both the N- and C-terminal domains of the protein. Cryo-trapping and kinetic measurements were used to probe the extent and temporal properties of protein motions for individual steps during photoconversion of Cph1. Formation of the primary photoproduct Lumi-R is not affected by changes in solvent viscosity and dielectric constant. Lumi-R formation occurs at cryogenic temperatures, consistent with their being no major structural reorganization of Cph1 during primary photoproduct formation. All remaining steps in the formation of the Pfr state are affected by solvent viscosity and dielectric constant and occur only at elevated temperatures, implying involvement of a series of long-range solvent-coupled conformational changes in Cph1. We show that signalling is achieved through ultrafast photoisomerization where localized structural change in the GAF domain is transmitted and amplified to cause larger-scale and slower conformational change in the PHY and histidine kinase domains. This hierarchy of timescales and extent of structural change orientates the histidine kinase domain to elicit the desired light-activated biological response.

Kinetic and spectroscopic probes of motions and catalysis in the cytochrome P450 reductase family of enzymes

There is a mounting body of evidence to suggest that enzyme motions are linked to function, although the design of informative experiments aiming to evaluate how this motion facilitates reaction chemistry is challenging. For the family of diflavin reductase enzymes, typified by cytochrome P450 reductase, accumulating evidence suggests that electron transfer is somehow coupled to large‐scale conformational change and that protein motions gate the electron transfer chemistry. These ideas have emerged from a variety of experimental approaches, including structural biology methods (i.e. X‐ray crystallography, electron paramagnetic/NMR spectroscopies and solution X‐ray scattering) and advanced spectroscopic techniques that have employed the use of variable pressure kinetic methodologies, together with solvent perturbation studies (i.e. ionic strength, deuteration and viscosity). Here, we offer a personal perspective on the importance of motions to electron transfer in the cytochrome P450 reductase family of enzymes, drawing on the detailed insight that can be obtained by combining these multiple structural and biophysical approaches.

The influence of the structure of the Au(110) surface on the ordering of a monolayer of cytochrome P450 reductase at the Au(110)/phosphate buffer interface

The reflection anisotropy spectra (RAS) observed initially from Au(110)/phosphate buffer interfaces at applied potentials of −0.652 and 0.056 V are very similar to the spectra observed from ordered Au(110) (1 × 3) and anion induced (1 × 1) surface structures respectively. These RAS profiles transform to a common profile after cycling the potential between these two values over 72 h indicating the formation of a less ordered surface. The RAS of a monolayer of a P499C variant of the human flavoprotein cytochrome P450 reductase adsorbed at 0.056 V at an ordered Au(110)/phosphate buffer interface is shown to arise from an ordered layer in which the optical dipole transitions are in a plane that is orientated roughly normal to the surface and parallel to either the [11̄0] or [001] axes of the Au(110) surface. The same result was found previously for adsorption of P499C on an ordered interface at −0.652 V. The adsorption of P499C at the disordered surface does not result in the formation of an ordered monolayer confirming that the molecular ordering is strongly influenced by both the local structure and the long range macroscopic order of the Au(110) surface.