Identification of a chloroplast coenzyme A-binding protein related to the peroxisomal thiolases.
Yang LiMing, Lamppa G.
Author Affiliation: Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA.
Plant Physiology 112 : 1641-1647
Abstract : A 30 kDa coenzyme A (CoA)-binding protein was isolated from spinach (Spinacia oleracea) cv. Melody chloroplast soluble extracts, using affinity chromatography such that 95% of the total protein was excluded. The 30 kDa protein contained an 8 amino acid sequence (DVRLYYGA) identical to a region in a 36 kDa protein of unknown function that is encoded by a kiwifruit (Actinidia deliciosa) cDNA. Southern blotting also detected a spinach gene related to this kiwifruit cDNA. The kiwifruit 36 kDa protein was synthesized in Escherichia coli, was imported into chloroplasts and cleaved to a 30 kDa form. The kiwifruit protein specifically bound to CoA and contained a single cysteine within a domain that was related to the peroxisomal ?-ketoacyl-CoA thiolases (which catalyse the CoA-dependent degradative step of fatty acid ?-oxidation). Within 50 amino acids surrounding the cysteine (considered to be part of the thiolase active site), the kiwifruit protein exhibited ~26% sequence identity with mango, cucumber and rat peroxisomal thiolases. N-terminal alignment with these enzymes, relative to the cysteine, indicated that the 36 kDa protein is cleaved after serine-58 during import, agreeing with the estimated size (approximately 6 kDa) of a transit peptide. The 30 kDa protein was also related to the E. coli and mitochondrial thiolases, as well as to the acetoacetyl-CoA thiolases of prokaryotes. Other features distinguish it from members of the thiolase family, and it is therefore suggested that it may carry out a related, but novel, function. It is suggested that the protein is more distantly related to chloroplast ?-ketoacyl-acyl carrier protein synthase III, the initial condensing enzyme of fatty acid synthetase that utilizes acetyl-CoA.