The glyoxysomal 3-ketoacyl-CoA thiolase precursor from Brassica napus has enzymatic activity when synthesized in Escherichia coli.
Glyoxysomal 3-ketoacyl-CoA thiolase is the last enzyme in the beta-oxidation of fatty acids in plant glyoxysomes. A full-length cDNA of the glyoxysomal 3-ketoacyl-CoA thiolase from Brassica napus and a truncated version, lacking the N-terminal targeting signal were cloned in a T7 promoter-based vector. Both recombinant proteins were expressed in Escherichia coli and activity was measured. Full-length and truncated 3-ketoacyl-CoA thiolase have comparable activity in E. coli. Moreover, full-length 3-ketoacyl-CoA thiolase was purified from E. coli and N-terminal sequencing of the protein confirmed that the precursor form indeed is enzymatically active.
Olesen
C
C
Department of Physiology, Carlsberg Laboratory, Copenhagen Valby, Denmark.
Thomsen
K K
KK
Svendsen
I
I
Brandt
A
A
D000102:Acetyl-CoA C-Acyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001937:Brassica / Q000201:enzymology*; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005227:Fatty Acids / Q000378:metabolism; D015870:Gene Expression; D007522:Isocitrate Lyase / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000378:metabolism; D008292:Malate Synthase / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000235:genetics; D011994:Recombinant Proteins; D012995:Solubility
D004792:Enzyme Precursors; D005227:Fatty Acids; D010446:Peptide Fragments; D011994:Recombinant Proteins; D008291:Malate Dehydrogenase; D000102:Acetyl-CoA C-Acyltransferase; D008292:Malate Synthase; D007522:Isocitrate Lyase