RESUMEN
Analysis of the beta-ketoacyl-ACP synthase (KAS) encoded by the fabF gene of Escherichia coli has been hampered by a reported instability of the cloned gene. Here we describe biochemical characterization of purified, active protein from the recombinant fabF gene. This enzyme has the properties ascribed to KAS II and not those of a putative KAS IV reported to be encoded by fabJ, a genomic clone with DNA sequence identical to that of fabF. We also characterize active protein from a recombinant fabB gene and suggest that this method may have a general utility for analysis of KAS enzymes.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Escherichia coli/enzimología , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/aislamiento & purificación , Cerulenina/farmacología , Clonación Molecular , Escherichia coli/genética , Genes Bacterianos , Vectores Genéticos , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , TemperaturaRESUMEN
Immature coconut (Cocos nucifera) endosperm contains a 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) activity that shows a preference for medium-chain-length fatty acyl-coenzyme A substrates (H.M. Davies, D.J. Hawkins, J.S. Nelsen [1995] Phytochemistry 39:989-996). Beginning with solubilized membrane preparations, we have used chromatographic separations to identify a polypeptide with an apparent molecular mass of 29 kD, whose presence in various column fractions correlates with the acyltransferase activity detected in those same fractions. Amino acid sequence data obtained from several peptides generated from this protein were used to isolate a full-length clone from a coconut endosperm cDNA library. Clone pCGN5503 contains a 1325-bp cDNA insert with an open reading frame encoding a 308-amino acid protein with a calculated molecular mass of 34.8 kD. Comparison of the deduced amino acid sequence of pCGN5503 to sequences in the data banks revealed significant homology to other putative LPAAT sequences. Expression of the coconut cDNA in Escherichia coli conferred upon those cells a novel LPAAT activity whose substrate activity profile matched that of the coconut enzyme.