WILD-TYPE AND MUTANT HUMAN HEART (R)-3-HYDROXYBUTYRATE DEHYDROGENASE EXPRESSED IN INSECT CELLS

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitocho ndrial enzyme with a specific requirement of phosphatidylcholine (PC) for function. PC is an allosteric activator that enhances NAD(H) bindi ng to BDH. The enzyme serves as a paradigm to study specific lipid-pro tein interactions in membranes. Analysis of the primary sequence of BD H, as determined by molecular cloning, predicts that lipid binding and substrate specificity are contributed by the C-terminal third of the protein [Marks. A. R., McIntyre, J. O., Duncan, T, M., Erdjument-Broma ge, H., Tempst. P., & Fleischer, S. (1992) J. Biol. Chem. 267, 15459-1 5463]. The mature form of human heart BDH has now been expressed in ca talytically active form in insect cells (Sf 9, Spodoptera frugiperda) transfected with BDH-cDNA in baculovirus. Endogenous PC in tile insect cells fulfills the lipid requirement for the expressed BDH since enzy matic activity is lost upon digestion with phospholipase A, and restor ed selectively by reconstitution with PC vesicles. The K(m)s for NAD() and (R)-3-hydroxybutyrate (R-HOB) of expressed BDH are similar to th ose for bovine heart or rat liver BDH in mitochondria. Replacing Cys24 2, (the only cysteine in the C-terminal domain) with serine by site-di rected mutagenesis resulted in a 10-fold increase in K-m for R-HOB wit h no change in the K-m for NAD(+), indicating a role for Cys242 in sub strate binding. Carboxypeptidase cleavage studies had indicated a requ irement of the C-terminal for catalysis and a role in lipid binding [A dami, P., Duncan, T. M., McIntyre, J. O., Carter, C. E., Fu, C., Melin , M., Latruffe, N., & Fleischer, S. (1993) Biochem J. 292, 863-872]. W e now show that deletion of twelve C-terminal amino acids to form a tr uncated BDH mutant results in loss of enzymic function. The expression in Sf9 cells of the constitutively active full-length mature form of human heart BDH and the first expression and characterization of BDH m utants validate this system for structure-function studies of BDH.