Oligomerization of Hydroperoxide Lyase, a Novel P450 Enzyme in Plants

Abstract

The oligomeric state of fatty acid hydroperoxide lyase (HPL), of molar mass ∼55 kDa is uncertain and it has been reported as a trimer or tetramer in vivo. The enzyme has been found to be bi-functional and is active even in the absence of detergent. The association with detergent is known to stabilise the binding of the enzyme to its substrate and the enzyme is more active. No high resolution structure of any plant P450 is available so far because of difficulty in crystallising the protein. We employ analytical ultracentrifugation to characterise the oligomeric state of an E. coli-expressed recombinant HPL from Medicago truncatula (HPL-F) under different solution conditions. Sedimentation velocity analyses show that HPL-F (under detergent-free conditions) is largely a monomer with a sedimentation coefficient s_20,w of ∼4.1 S (a value expected from the molar mass of the monomer). The effects of protein concentration, and detergent micelles on the oligomeric state of detergent-free HPL-F are reported for the first time. With increase in protein concentration only traces of dimers can be detected. However, HPL-F in association with detergent is a mixture of oligomers, which are not in reversible equilibrium with each other. These studies have important implications as they show that the oligomeric state of HPL-F changes with micellar association, both of which are related to the activity of the protein. They also show the virtue of combining sedimentation velocity with sedimentation equilibrium in the ultracentrifuge for the study of enzyme-detergent systems.