Local Conformational Constraint of Firefly Luciferase Can Affect the Energy of Bioluminescence and Enzyme Stability

Conformational dynamics contribute importantly to enzyme catalysis,such that targeted conformational constraint may affect catalysis.Firefly luciferases undergo extensive structural change during catalysis;key residues form a hydrophobic pocket,excluding water and enabling maximally energetic light production.Point mutants almost always luminesce at longer wavelengths(lower energy)than the wild type.Conformational constraint,using dipeptide analogue 3 at a position critical for optimized excited state structure,produced luciferase emission at a shorter wavelength by∼10 nm.Incomparison,introduction of conformationally constrained analogues 4,5,or 7 afforded luciferases emitting at longer wavelengths,while a related unconstrained luciferase(analogue 6)exhibited wild-type emission.The constrained luciferases tested were more stable than the wild type.Protein modeling demonstrated that the“inside”or“outside”orientation of the conformationally constrained dipeptide led to the shorter or longer emission wavelength,respectively.More broadly,these results suggest that local conformational constraint can control specific elements of enzyme behavior,both in vitro and in vivo.This represents the first example of studying enzyme function by introducing conformationally constrained dipeptides at a specific protein position.The principles discovered here in luciferase modification will enable studies to control the wavelength emission and photophysical properties of modified luciferases.

Selective epoxidation of linear terminal olefins with metalloporphyrins under mild conditions

The epoxidation of linear terminal olefins with metalloporphyrins in the presence of dioxygen and isobutyraldehyde under ambient temperature and atmo-spheric pressure was investigated.The results show that all olefins could be smoothly converted to epoxides with high selectivities(70%–90%).For the metalloporphyrins with different catalytic activities within 1-hexene epoxidation in the order of Fe>Mn>Co,T(o-Cl)PPFe(III)Cl was most effective,with a 41.7%yield and 80.2%selectivity of 1,2-epoxyhexane.Various amounts of catalyst were investi-gated,and it was found that with only 10ppm catalyst the yield of 1,2-epoxyhexane and turnover number(TON)could reach up to 41.9%and 41859,respectively.