Lipid Vesicles Charges Determine Dehydrogenase Vs Isomerase Activity

Presentation Number: MON 515
Date of Presentation: April 3rd, 2017

Maheshinie Rajapaksha*1, James L Thomas2 and Himangshu S Bose3
1Mercer University, Savannah, GA, 2Mercer University School of Medicine, Macon, GA, 3Mercer University School of Medicine, savannah, GA

Abstract

Steroid hormones are essential for mammalian survival, and a key early step in biosynthetic pathway starts at the matrix side of mitochondria. Mitochondria is one of the smallest organelle in the cell, and it has four subcompartments; Outer membrane (OMM), Intermembranous space (IMS), Inner membrane (IMM), and the matrix. IMS resident, 3beta-hydroxysteroid dehydrogenase (3βHSD2), catalyzes the conversion of pregnenolone (Preg) to progesterone (Prog) and of dehydroepiandrosterone (DHEA) to androstenedione (AD) using the dehydrogenase and isomerase activities of the bifunctional enzyme. Genetic mutations in 3βHSD2 result in hypertension, salt wasting crisis and impaired sexual development. The mechanism of dual functionality of 3βHSD2 related to dehydrogenase and isomerase activities at the IMS is still not clearly understood. We hypothesize that 3βHSD2 associates first with the IMS of OMM and then to the IMM of lipid membrane by flipping from one conformational state to the next one with the help of charged lipid membrane. Here we report that in the presence of phosphocholine (PC), 3βHSD2 converted Preg to Prog with a 4-fold increase in synthesis. In the presence of negatively charged cardiolipin (CL), Preg to Prog synthesis reduced to the base level. However, under similar condition AD synthesis was increased more than 2 fold. Conformational analysis by circular dichroism showed stabilization of the protein conformation with the OMM lipid composition. Using fluorescence spectrophotometry (FRET), we found that 3βHSD2 binds to both PC and CL under equilibrium conditions where the energetic equilibrium remained unchanged. This observation supports the idea that lipids associate with 3βHSD2 to form a transient and active complex with two different confirmations. So, we conclude that lipid vesicles assist 3βHSD2 to flip between confirmations to rapidly produce steroids when needed depending on the equilibrium condition between these two conformations.

 

Nothing to Disclose: MR, JLT, HSB