Saltar al contenido
Merck

Production and characterization of recombinant perdeuterated cholesterol oxidase.

Analytical biochemistry (2015-06-16)
Emily Golden, Paul V Attwood, Anthony P Duff, Flora Meilleur, Alice Vrielink
RESUMEN

Cholesterol oxidase (CO) is a FAD (flavin adenine dinucleotide) containing enzyme that catalyzes the oxidization and isomerization of cholesterol. Studies directed toward elucidating the catalytic mechanism of CO will provide an important general understanding of Flavin-assisted redox catalysis. Hydrogen atoms play an important role in enzyme catalysis; however, they are not readily visualized in protein X-ray diffraction structures. Neutron crystallography is an ideal method for directly visualizing hydrogen positions at moderate resolutions because hydrogen and deuterium have comparable neutron scattering lengths to other heavy atoms present in proteins. The negative coherent and large incoherent scattering lengths of hydrogen atoms in neutron diffraction experiments can be circumvented by replacing hydrogen atoms with its isotope, deuterium. The perdeuterated form of CO was successfully expressed from minimal medium, purified, and crystallized. X-ray crystallographic structures of the enzyme in the perdeuterated and hydrogenated states confirm that there are no apparent structural differences between the two enzyme forms. Kinetic assays demonstrate that perdeuterated and hydrogenated enzymes are functionally identical. Together, structural and functional studies indicate that the perdeuterated protein is suitable for structural studies by neutron crystallography directed at understanding the role of hydrogen atoms in enzyme catalysis.

MATERIALES
Referencia del producto
Marca
Descripción del producto

Sigma-Aldrich
Imidazol, ReagentPlus®, 99%
Sigma-Aldrich
Imidazol, for molecular biology, ≥99% (titration)
Sigma-Aldrich
Imidazole buffer Solution, BioUltra, 1 M in H2O
Sigma-Aldrich
Imidazol, ACS reagent, ≥99% (titration)
Sigma-Aldrich
Imidazol, BioUltra, ≥99.5% (GC)
Sigma-Aldrich
Imidazol, puriss. p.a., ≥99.5% (GC)
Sigma-Aldrich
Imidazol, ≥99% (titration), crystalline
Sigma-Aldrich
Imidazol, BioUltra, for molecular biology, ≥99.5% (GC)
Sigma-Aldrich
Glycerol-1,1,2,3,3-d5, 98 atom % D
Sigma-Aldrich
Imidazol, anhydrous, free-flowing, Redi-Dri, ACS reagent, ≥99%
Sigma-Aldrich
Glycerol-1,1,2,3,3-d5, endotoxin tested, 98 atom % D
Sigma-Aldrich
Imidazol, ReagentPlus®, 99%, Redi-Dri, free-flowing
Sigma-Aldrich
Imidazol, for molecular biology, ≥99% (titration), free-flowing, Redi-Dri