Identification of a chloroplast coenzyme A-binding protein related to the peroxisomal thiolases.
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.
Yang
L M
LM
Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637, USA.
Lamppa
G
G
D000101:Acetyl-CoA C-Acetyltransferase / Q000378:metabolism*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D003065:Coenzyme A / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008830:Microbodies / Q000201:enzymology*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D000101:Acetyl-CoA C-Acetyltransferase; D003065:Coenzyme A