Cyclooxygenases are dual-function heme proteins critical for the biosynthesis of prostaglandins in mammals. Mammalian cyclooxygenases contain both dioxygenase activity (adding molecular oxygen to lipid substrates to form lipid hydroperoxides) and peroxidase activity (reducing the hydroperoxides to alcohols). Two open reading frames in the ammonia-oxidizing betaproteobacterium Nitrosospira multiformis are annotated as cyclooxygenases by structural prediction of the protein product. We have attempted to express and characterize one of these open reading frames to test the structural prediction. DNA encoding the desired open reading frame was synthesized and inserted into several expression vectors with N-terminal fusion partners (his6 tag, SUMO, thioredoxin). Protein expression yielded largely insoluble protein product under many different expression conditions. Recently, we have been able to express a thioredoxin-Nitrosospira cyclooxygenase fusion partner in soluble form. Ongoing experiments to characterize the protein structurally and enzymatically will be reported.
