Yang L M, Lamppa G
Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637, USA.
Plant Physiol. 1996 Dec;112(4):1641-7. doi: 10.1104/pp.112.4.1641.
A 30-kD coenzyme A (CoA)-binding protein was isolated from spinach (Spinacea oleracea) chloroplast soluble extracts using affinity chromatography under conditions in which 95% of the total protein was excluded. The 30-kD protein contains an eight-amino-acid sequence, DVRLYYGA, that is identical to a region in a 36-kD protein of unknown function that is encoded by a kiwifruit (Actinidia deliciosa) cDNA. Southern blotting also detected a spinach gene that is related to the kiwifruit cDNA. The kiwifruit 36-kD protein that was synthesized in Escherichia coli was imported into chloroplasts and cleaved to a 30-kD form; it was processed to the same size in an organelle-free assay. Furthermore, the kiwifruit protein specifically bound to CoA. The kiwifruit protein contains a single cysteine within a domain that is related to the peroxisomal beta-ketoacyl-CoA thiolases, which catalyze the CoA-dependent degradative step of fatty acid beta-oxidation. Within 50 amino acids surrounding the cysteine, considered to be part of the thiolase active site, the kiwifruit protein shows approximately 26% sequence identity with the mango, cucumber, and rat peroxisomal thiolases. N-terminal alignment with these enzymes, relative to the cysteine, indicates that the 36-kD protein is cleaved after serine-58 during import, agreeing with the estimated size (approximately 6 kD) of a transit peptide. The 30-kD protein is also related to the E. coli and mitochondrial thiolases, as well as to the acetoacetyl-CoA thiolases of prokaryotes. Features distinguish it from members of the thiolase family, suggesting that it carries out a related but novel function. The protein is more distantly related to chloroplast beta-ketoacyl-acyl carrier protein synthase III, the initial condensing enzyme of fatty acid synthetase that utilizes acetyl-CoA.
利用亲和层析法，在排除95%总蛋白的条件下，从菠菜（Spinacea oleracea）叶绿体可溶性提取物中分离出一种30-kD辅酶A（CoA）结合蛋白。该30-kD蛋白包含一个八氨基酸序列DVRLYYGA，与由猕猴桃（Actinidia deliciosa）cDNA编码的未知功能的36-kD蛋白中的一个区域相同。Southern印迹法也检测到菠菜中一个与猕猴桃cDNA相关的基因。在大肠杆菌中合成的猕猴桃36-kD蛋白被导入叶绿体并被切割成30-kD形式；在无细胞器的分析中它被加工成相同大小。此外，猕猴桃蛋白特异性结合CoA。猕猴桃蛋白在与过氧化物酶体β-酮酰基辅酶A硫解酶相关的结构域内含有一个半胱氨酸，该酶催化脂肪酸β-氧化的CoA依赖性降解步骤。在被认为是硫解酶活性位点一部分的半胱氨酸周围的50个氨基酸内，猕猴桃蛋白与芒果、黄瓜和大鼠过氧化物酶体硫解酶显示出约26%的序列同一性。相对于半胱氨酸与这些酶的N端比对表明，36-kD蛋白在导入过程中在丝氨酸-58之后被切割，这与转运肽的估计大小（约6 kD）一致。30-kD蛋白也与大肠杆菌和线粒体硫解酶以及原核生物的乙酰乙酰辅酶A硫解酶相关。其特征使其与硫解酶家族成员不同，表明它执行相关但新颖的功能。该蛋白与叶绿体β-酮酰基-酰基载体蛋白合酶III的关系更远，后者是利用乙酰辅酶A的脂肪酸合成酶的初始缩合酶。
