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首页> 外文期刊>Biochemistry >WILD TYPE AND MUTANT HUMAN HEART (R)-3-HYDROXYBUTYRATE DEHYDROGENASE EXPRESSED IN INSECT CELLS
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WILD TYPE AND MUTANT HUMAN HEART (R)-3-HYDROXYBUTYRATE DEHYDROGENASE EXPRESSED IN INSECT CELLS

机译：在昆虫细胞中表达的野生型和突变型人类心脏（R）-3-羟基丁酸脱氢酶

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摘要

(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 (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 enzymatic activity is lost upon digestion with phospholipase A, 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 Sf9 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.
机译：（R）-3-羟基丁酸脱氢酶（BDH）是一种需要脂质的线粒体酶，对磷脂酰胆碱（PC）的功能有特殊要求。 PC是一种变构激活剂，可增强NAD（H）与BDH的结合。该酶充当研究膜中特定脂蛋白相互作用的范例。通过分子克隆确定的对BDH一级序列的分析预测，脂质结合和底物特异性是由蛋白质的C末端三分之一贡献的。 A. R.，McIntyre，J. O.，Duncan，T，M.，Erdjument-Bromage，H.，Tempst。 P.和Fleischer，S。（1992）生物化学杂志。化学267，15459-15463]。现在，人类心脏BDH的成熟形式已经在杆状病毒中以BDH-cDNA转染的昆虫细胞（Sf 9，Spodoptera frugiperda）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的脂质需求，因为在用磷脂酶A消化后会失去酶促活性，并通过与PC囊泡的重构选择性恢复。表达的BDH的NAD（+）和（R）-3-羟基丁酸（R-HOB）的K（m）与线粒体中牛心或大鼠肝脏BDH的K（m）相似。通过位点诱变用丝氨酸取代Cys242（C端结构域中唯一的半胱氨酸），导致R-HOB的Km升高10倍，而NAD（+）的Km不变，表明其作用Cys242与底物结合。羧肽酶裂解研究表明，催化作用需要C末端，并且在脂质结合中发挥作用[Adami，P.，Duncan，TM，McIntyre，JO，Carter，CE，Fu，C.，Melin，M.，Latruffe， N。，＆Fleischer，S。（1993）Biochem J.292，863-872]。我们现在显示，删除12个C末端氨基酸以形成截短的BDH突变体会导致酶功能丧失。人心脏BDH的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用于BDH的结构功能研究。

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