COx from Brevibacterium Sterolicum.
a) Non-covalently bound FAD;
b) covalently-bound FAD
Movie of oxidation/isomerization mechanism (QuickTime App Download)
b) covalently-bound FAD
Movie of oxidation/isomerization mechanism (QuickTime App Download)
We are studying two different forms of cholesterol oxidase from Brevibacterium sterolicum, one which contains the FAD cofactor non-covalently bound to the enzyme and one in which the cofactor is covalently linked to a histidine residue. Interestingly these two different forms of cholesterol oxidase, found in the same bacterial species, do not share any significant sequence homology, despite the fact that they carry out identical catalytic reactions. Furthermore, the difference in the redox potentials for these two forms of the enzyme is ~150 mV suggesting that the protein structure in the region of the cofactor has a significant effect on the redox activity exhibited by the cofactor. We have determined the crystal structures of both enzymes and found them to be very different from each other (see figures) including significant differences in the active sites. These differences suggest that the two enzymes undergo different catalytic mechanisms. We are currently comparing the two different catalytic mechanisms by a combination of site directed mutagenesis, kinetic analysis and further structural studies.
We have also solved the structure of another cholesterol oxidase (from Streptomyces sp.) and refined it to 0.95A. The high resolution structure reveals some surprising features, such as protonation of the NE2 atom of H477 (which until now was thought to act as a base in the redox reaction) and a narrow tunnel that we propose allows access of molecular oxygen to the active site. Currently, we are attempting to obtain diffraction data at even higher resolution, which would reveal a detailed electron topography of the active site. Structural studies of the flavoenzyme active site at subatomic resolution would greatly enhance our knowledge of redox catalysis and improve the existing theoretical calculations.
L-amino acid oxidase | Cholesterol oxidase | Formiminotransferase-cyclodeaminase (FTCD)
DmpFG | LuxCDE
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