A new approach to understanding structure-function relationships in cytochromes P450 by targeting terpene metabolism in the wild.

A strategy for elucidating sequence determinants of function in the class of cytochrome P450 (CYP) enzymes that catalyze the first steps of terpene metabolism in wild microbiomes is described. Wild organisms that can use camphor, terpineol, pinene and limonene were isolated from soils rich in coniferous waste. Cell free extracts and growth beers were analyzed by gas chromatography/mass spectrometry to identify primary oxidative metabolites.

Substrate recognition by two different P450s: Evidence for conserved roles in a common fold.

Cytochrome P450 monooxygenases CYP101A1 and MycG catalyze regio- and stereospecific oxidations of their respective substrates, d-camphor and mycinamicin IV. Despite the low sequence homology between the two enzymes (29% identity) and differences in size and hydrophobicity of their substrates, the conformational changes that occur upon substrate binding in both enzymes as determined by solution NMR methods show some striking similarities. Many of the same secondary structural features in both enzymes are perturbed, suggesting the existence of a common mechanism for substrate binding and recognition in the P450 superfamily.

NADH reduction of nitroaromatics as a probe for residual ferric form high-spin in a cytochrome P450.

The existence of a substrate-sensitive equilibrium between high spin (S=5/2) and low spin (S=1/2) ferric iron is a well-established phenomenon in the cytochrome P450 (CYP) superfamily, although its origins are still a subject of discussion. A series of mutations that strongly perturb the spin state equilibrium in the camphor hydroxylase CYP101A1 were recently described (Colthart et al., Sci.

Detection of substrate-dependent conformational changes in the P450 fold by nuclear magnetic resonance.

Cytochrome P450 monooxygenases typically catalyze the insertion of one atom of oxygen from O2 into unactivated carbon-hydrogen and carbon-carbon bonds, with concomitant reduction of the other oxygen atom to H2O by NAD(P)H. Comparison of the average structures of the camphor hydroxylase cytochrome P450(cam) (CYP101) obtained from residual dipolar coupling (RDC)-restrained molecular dynamics (MD) in the presence and absence of substrate camphor shows structural displacements resulting from the essential collapse of the active site upon substrate removal. This collapse has conformational consequences that extend across the protein structure, none of which were observed in analogous crystallographic structures.

Enabling the synthesis of perfluoroalkyl bicyclobutanes via 1,3 γ-silyl elimination.

Two new bicyclobutanes were prepared from cyclobutyl systems by a novel, solvolytic, carbocation-based methodology. An electron-withdrawing perfluoroalkyl group at the incipient cationic center enhances neighboring-group participation of the γ-silyl group, inducing facile, remarkably selective 1,3-elimination yielding only bicyclobutanes. The method unlocks potential access to a host of EWG-substituted strained rings and a potential new method for the synthesis of trifluoromethylcyclopropanes.