3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus).
3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.
Sheldon
P S
PS
Department of Biochemistry, University of Birmingham, Edgbaston, UK.
Kekwick
R G
RG
Smith
C G
CG
Sidebottom
C
C
Slabas
A R
AR
D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001937:Brassica / Q000201:enzymology*; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D064429:Fatty Acid Synthases / Q000737:chemistry* / Q000378:metabolism; D007150:Immunohistochemistry; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D016415:Sequence Alignment
D000596:Amino Acids; D046911:Macromolecular Substances; D000429:Alcohol Oxidoreductases; D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D064429:Fatty Acid Synthases