Luet Lok Wong

A highly active single-mutation variant of P450BM3 (CYP102A1)

The power of proline: Bold amino acid substitutions in sensitive protein regions are frequently unproductive, while more subtle mutations can be sufficient to bring about dramatic changes. But introducing proline at the residue next to the sulfur ligand in P450BM3 (CYP102A1) has the unexpected and desirable effect of enhancing the activity of this fatty acid hydroxylase with a broad range of non‐natural substrates, as illustrated by the figure.

The structure of CYP101D2 unveils a potential path for substrate entry into the active site

The cytochrome P450 CYP101D2 from Novosphingobium aromaticivorans DSM12444 is closely related to CYP101D1 from the same bacterium and to P450cam (CYP101A1) from Pseudomonas putida. All three are capable of oxidizing camphor stereoselectively to 5-exo-hydroxycamphor. The crystal structure of CYP101D2 revealed that the likely ferredoxin-binding site on the proximal face is largely positively charged, similar to that of CYP101D1. However, both the native and camphor-soaked forms of CYP101D2 had open conformations with an access channel. In the active site of the camphor-soaked form, the camphor carbonyl interacted with the haem-iron-bound water. Two other potential camphor-binding sites were also identified from electron densities in the camphor-soaked structure: one located in the access channel, flanked by the B/C and F/G loops and the I helix, and the other in a cavity on the surface of the enzyme near the F helix side of the F/G loop. The observed open structures may be conformers of the CYP101D2 enzyme that enable the substrate to enter the buried active site via a conformational selection mechanism. The second and third binding sites may be intermediate locations of substrate entry and translocation into the active site, and provide insight into a multi-step substrate-binding mechanism.

Drug Oxidation by Cytochrome P450BM3: Metabolite Synthesis and Discovering New P450 Reaction Types

There is intense interest in late-stage catalytic C-H bond functionalization as an integral part of synthesis. Effective catalysts must have a broad substrate range and tolerate diverse functional groups. Drug molecules provide a good test of these attributes of a catalyst. A library of P450BM3 mutants developed from four base mutants with high activity for hydrocarbon oxidation produced human metabolites of a panel of drugs that included neutral (chlorzoxazone, testosterone), cationic (amitriptyline, lidocaine) and anionic (diclofenac, naproxen) compounds. No single mutant was active for all the tested drugs but multiple variants in the library showed high activity with each compound. The high conversions enabled full product characterization that led to the discovery of the new P450 reaction type of oxidative decarboxylation of an α-hydroxy carboxylic acid and the formation a protected imine from an amine, offering a novel route to α-functionalization of amines. The substrate range and varied product profiles suggest that this library of enzymes is a good basis for developing late-stage C-H activation catalysts.

Characterisation of the paramagnetic [2Fe–2S]+ centre in palustrisredoxin-B (PuxB) from Rhodopseudomonas palustris CGA009: g-matrix determination and spin coupling analysis

Palustrisredoxin-B (PuxB) from Rhodopseudomonas palustris (CGA009) is a [2Fe-2S] ferredoxin which is able to accept electrons from NADH via the flavin-dependent palustrisredoxin reductase (PuR); these electrons can then be transferred to the P450 enzyme (CYP199A2). This work reports on the paramagnetic state of the [2Fe-2S](+) cluster in PuxB, both alone and in the PuR-PuxB complex. Aided by the X-ray crystal structure of PuxB, the protons nearest to the reduced [2Fe-2S](+) cluster were used as magnetic probes to quantify the g-matrix orientation and anisotropic magnetic moment of the paramagnetic centre. (1)H hyperfine couplings were measured with W-band Davies ENDOR and X-band HYSCORE spectroscopy and fitted to a model in which (1)H dipolar couplings were calculated assuming point magnetic moments located at the Fe ions, and bridging and coordinating cysteine sulfur atoms. The absolute sign of a (1)H hyperfine coupling was measured using a variable mixing time ENDOR experiment to confirm the assignment of the Fe(3+) and Fe(2+) ions. For the anti-ferromagnetically coupled cluster the magnetic moment is described in terms of spin projection factors, and our analysis yields values of K(exp)(A) = +2.33 to +1.85 (ferric site), and K(exp)(B) = -1.33 to -0.85 (ferrous site). These values are discussed in terms of the delocalisation of the spin density and hence the limitations of applying a local site spin coupling model to calculate the spin projection factors in a complex with considerable overlap of the α- and β-spin magnetic oribitals. The accurate description of the g-matrix orientation and magnetic moment of this [2Fe-2S](+) cluster enable it to be utilised as a paramagnetic spin probe, for example, to measure electron-electron distances. In the pdb reference frame of PuxB (code ) the g(∥) axis vector is g(∥) = [-0.6524 ± 0.0248, -0.6269 ± 0.0115, 0.4259 ± 0.0405], with the principal g-values of g(⊥) = 1.9328 ± 0.0003, g(∥) = 2.0233 ± 0.0003.

A new mechanism for exchange processes observed in the compounds [M(η-C5H5)2(exo-η-RCHCH2)H], M = Nb and Ta

Dynamic n.m.r. studies of the exchange processes in the complexes [M(η-C_5H_5)(exo-η-RCH=CH_2)H], M = Nb, Ta, lead to the proposal of a new mechanism involving intermediates with agostic bonding.

A Structural Model of a P450-Ferredoxin Complex from Orientation-Selective Double Electron-Electron Resonance Spectroscopy

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of chemically inert carbon-hydrogen bonds in diverse endogenous and exogenous organic compounds by atmospheric oxygen. This C-H bond oxy-functionalization activity has huge potential in biotechnological applications. Class I CYPs receive the two electrons required for oxygen activation from NAD(P)H via a ferredoxin reductase and ferredoxin. The interaction of Class I CYPs with their cognate ferredoxin is specific. In order to reconstitute the activity of diverse CYPs, structural characterization of CYP-ferredoxin complexes is necessary, but little structural information is available. Here we report a structural model of such a complex (CYP199A2-HaPux) in frozen solution derived from distance and orientation restraints gathered by the EPR technique of orientation-selective double electron-electron resonance (os-DEER). The long-lived oscillations in the os-DEER spectra were well modeled by a single orientation of the CYP199A2-HaPux complex. The structure is different from the two known Class I CYP-Fdx structures: CYP11A1-Adx and CYP101A1-Pdx. At the protein interface, HaPux residues in the [Fe2S2] cluster-binding loop and the α3 helix and the C-terminus residue interact with CYP199A2 residues in the proximal loop and the C helix. These residue contacts are consistent with biochemical data on CYP199A2-ferredoxin binding and electron transfer. Electron-tunneling calculations indicate an efficient electron-transfer pathway from the [Fe2S2] cluster to the heme. This new structural model of a CYP-Fdx complex provides the basis for tailoring CYP enzymes for which the cognate ferredoxin is not known, to accept electrons from HaPux and display monooxygenase activity.

Direct electrochemistry of pentachlorophenol hydroxylase

The direct electrochemistry of flavin-containing monooxygenase, pentachlorophenol hydroxlase (PCPH), has been investigated under a variety of conditions. PCPH underwent a two-electron process on the electrodes, which correspond to the reduction/oxidation of FAD/FADH2 within the enzyme. The electrochemical response of PCPH relies on the interface of the electrode and the enzyme solution. Three types of interaction of the enzyme molecule with the electrodes were observed: at a bare edge-plane graphite electrode, diffusion-controlled process was observed, suggesting the interaction is weak. In the presence of the cations, the interaction became stronger, so that the voltammetric response changed from a diffusion control to an adsorption control An intermediate case was observed at a poly(L- lysine) modified EPG electrode.