title: Beta-ketoacyl-acyl carrier protein synthase III from pea (Pisum sativum L.): properties, inhibition by a novel thiolactomycin analogue and isolation of a cDNA clone encoding the enzyme.
abstract: A beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III; short-chain condensing enzyme) has been partly purified from pea leaves. The enzyme, which had acetyl-CoA:ACP acyltransferase (ACAT) activity, was resolved from a second, specific, ACAT protein. The KAS III enzyme had a derived molecular mass of 42 kDa (from its cDNA sequence) and operated as a dimer. Its enzymological characteristics were similar to those of two other plant KAS III enzymes except for its inhibition by thiolactomycin. A derivative of thiolactomycin containing a longer (C8 saturated) hydrophobic side-chain (compound 332) was a more effective inhibitor of pea KAS III and showed competitive inhibition towards malonyl-ACP whereas thiolactomycin showed uncompetitive characteristics at high concentrations. This difference may be due to the better fit of compound 332 into a hydrophobic pocket at the active site. A full-length cDNA for the pea KAS III was isolated. This was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in order to facilitate subsequent purification. Demonstrated activity in preparations from E. coli confirmed that the cDNA encoded a KAS III enzyme. Furthermore, the expressed KAS III had ACAT activity, showing that the latter was inherent. The derived amino acid sequence of the pea cDNA showed 81-87% similarity to that for other plant dicotyledon KAS IIIs, somewhat less for Allium porrum (leek, 71%) and for Porphyra spp. (62%), Synechocystis spp. (65%) and various bacteria (42-65%). The pea KAS III exhibited four areas of homology, three of which were around the active-site Cys(123), His(323) and Asn(353). In addition, a stretch of 23 amino acids (residues 207-229 in the pea KAS III) was almost completely conserved in the plant KAS IIIs. Modelling this stretch showed they belonged to a peptide fragment that fitted over the active site and contained segments suggested to be involved in substrate binding and in conformational changes during catalysis, as well as an arginine suggested to participate in the acid-base catalytic mechanism.
authors.affiliation: School of Biosciences, Cardiff University, PO Box 911, Cardiff, CF10 3US, UK.
authors.forename: A Lesley
authors.initials: AL
authors.lastname: Jones
authors.forename: Andy M
authors.initials: AM
authors.lastname: Gane
authors.forename: Derek
authors.initials: D
authors.lastname: Herbert
authors.forename: David L
authors.initials: DL
authors.lastname: Willey
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Rutter
authors.forename: Peter
authors.initials: P
authors.lastname: Kille
authors.forename: Jane E
authors.initials: JE
authors.lastname: Dancer
authors.forename: John L
authors.initials: JL
authors.lastname: Harwood
mesh_terms: D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D015256:Coenzyme A-Transferases / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology / Q000235:genetics*; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D013876:Thiophenes / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D018076:DNA, Complementary; D013876:Thiophenes; C035068:thiolactomycin; C077402:3-ketoacyl-acyl carrier protein synthase III; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D015256:Coenzyme A-Transferases; C026389:acetyl-CoA-acetoacetate CoA transferase