Characterization of the malonyl-/acetyltransacylase domain of the multifunctional animal fatty acid synthase by expression in Escherichia coli and refolding in vitro.
cDNAs of various lengths encoding the second domain of the multifunctional fatty acid synthase (FAS) have been expressed in Escherichia coli and the recombinant proteins refolded in vitro to catalytically active monomeric malonyl-/acetyltransacylases. FAS residues 428-487, previously thought to represent the amino terminus of the malonyl-/acetyltransacylase, can be omitted from the recombinant enzyme with no loss in catalytic activity. This shortened transacylase, consisting of FAS residues 488-809, can be repeatedly denatured and renatured in vitro with reproducibly high recovery and no loss in specific activity. When expressed as a soluble enzyme in Spodoptera frugiperda cells, this transacylase has the same specific activity as the enzyme that has been refolded in vitro. The refolded transacylase consisting of FAS residues 488-809, but not the longer enzyme consisting of residues 428-815, can be crystallized readily. These results suggest that FAS residues 428-487, previously thought to represent the amino terminus of the malonyl-/acetyltransacylase, are not required for catalysis of the transacylase reaction. This region of the FAS is less well conserved than the transacylase catalytic domain and may constitute an extended structural linker that facilitates the functional interaction between the transacylase and acyl carrier protein domains.
Rangan
V S
VS
Children's Hospital Oakland Research Institute, Oakland, CA 94609, USA.
Serre
L
L
Witkowska
H E
HE
Bari
A
A
Smith
S
S
D000102:Acetyl-CoA C-Acyltransferase / Q000235:genetics / Q000378:metabolism*; D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002478:Cells, Cultured; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D064429:Fatty Acid Synthases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D018411:Spodoptera; D014162:Transfection
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D000217:Acyltransferases; D000102:Acetyl-CoA C-Acyltransferase; D051080:Acyl-Carrier Protein S-Malonyltransferase; C494968:fabD protein, E coli; D064429:Fatty Acid Synthases; D054889:Fatty Acid Synthase, Type II