Green D, Marks A R, Fleischer S, McIntyre J O
Brookdale Center for Molecular Biology Mount Sinai School of Medicine, New York, New York 10032 USA.
Biochemistry. 1996 Jun 25;35(25):8158-65. doi: 10.1021/bi952807n.
(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme with a specific requirement of phosphatidylcholine (PC) for function. PC is an allosteric activator that enhances NAD(H) binding to BDH. The enzyme serves as a paradigm to study specific lipid-protein interactions in membranes. Analysis of the primary sequence of BDH, 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-Bromage, H., Tempst, P., & Fleischer, S. (1992) J. Biol. Chem. 267, 15459-15463]. The mature form of human heart BDH has now been expressed in catalytically active form in insect cells (Sf9, Spodoptera frugiperda) transfected with BDH-cDNA in baculovirus. Endogenous PC in the insect cells fulfills the lipid requirement for the expressed BDH since enzymatic activity is lost upon digestion with phospholipase A2 and restored selectively by reconstitution with PC vesicles. The K(m)s for NAD+ and (R)-3-hydroxybutyrate (R-HOB) of expressed BDH are similar to those for bovine heart or rat liver BDH in mitochondria. Replacing Cys242 (the only cysteine in the C-terminal domain) with serine by site-directed mutagenesis resulted in a 10-fold increase in K(m) for R-HOB with no change in the K(m) for NAD+, indicating a role for Cys242 in substrate binding. Carboxypeptidase cleavage studies had indicated a requirement of the C-terminal for catalysis and a role in lipid binding [Adami, P., Duncan, T. M., McIntyre, J. O., Carter, C. E., Fu, C., Melin, M., Latruffe, N., & Fleischer, S. (1993) Biochem J. 292, 863-872]. We now show that deletion of twelve C-terminal amino acids to form a truncated BDH mutant results in loss of enzymic function. The expression in Sf 9 cells of the constitutively active full-length mature form of human heart BDH and the first expression and characterization of BDH mutants validate this system for structure-function studies of BDH.
（R）-3-羟基丁酸脱氢酶（BDH）是一种需要脂质的线粒体酶，其功能对磷脂酰胆碱（PC）有特定需求。PC是一种变构激活剂，可增强NAD（H）与BDH的结合。该酶是研究膜中特定脂质-蛋白质相互作用的范例。通过分子克隆确定的BDH一级序列分析预测，脂质结合和底物特异性由蛋白质的C端三分之一决定[马克斯，A.R.，麦金太尔，J.O.，邓肯，T.M.，厄朱门特-布罗梅奇，H.，滕普斯特，P.，&弗莱舍尔，S.（1992）《生物化学杂志》267，15459 - 15463]。人心脏BDH的成熟形式现已在杆状病毒中用BDH - cDNA转染的昆虫细胞（Sf9，草地贪夜蛾）中以催化活性形式表达。昆虫细胞中的内源性PC满足了表达的BDH对脂质的需求，因为用磷脂酶A2消化后酶活性丧失，而通过用PC囊泡重构可选择性恢复。表达的BDH对NAD +和（R）-3-羟基丁酸（R - HOB）的米氏常数（K（m））与线粒体中牛心脏或大鼠肝脏BDH的相似。通过定点诱变将Cys242（C端结构域中唯一的半胱氨酸）替换为丝氨酸，导致R - HOB的K（m）增加10倍，而NAD +的K（m）不变，表明Cys242在底物结合中起作用。羧肽酶切割研究表明C端对催化有需求且在脂质结合中起作用[阿达米，P.，邓肯，T.M.，麦金太尔，J.O.，卡特，C.E.，傅，C.，梅林，M.，拉特吕夫，N.，&弗莱舍尔，S.（1993）《生物化学杂志》292，863 - 872]。我们现在表明，缺失十二个C端氨基酸以形成截短的BDH突变体导致酶功能丧失。人心脏BDH组成型活性全长成熟形式在Sf9细胞中的表达以及BDH突变体的首次表达和表征验证了该系统可用于BDH的结构-功能研究。
