CHARACTERIZATION OF THE MALONYLTRANSACYLASE ACETYLTRANSACYLASE DOMAINOF 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 multifuncti onal fatty acid synthase (FAS) have been expressed in Escherichia coil and the recombinant proteins refolded in vitro to catalytically activ e monomeric malonyl-/acetyltransacylases. FAS residues 428-487, previo usly thought to represent the amino terminus of the malonyl-/acetyltra nsacylase, can be omitted from the recombinant enzyme with no loss in catalytic activity, This shortened transacylase, consisting of FAS res idues 488-809, can be repeatedly denatured and renatured in vitro with reproducibly high recovery and no loss in specific activity, When exp ressed as a soluble enzyme in Spodoptera frugiperda cells, this transa cylase has the same specific activity as the enzyme that has been refo lded in vitro, The refolded transacylase consisting of FAS residues 48 8-809, but not the longer enzyme consisting of residues 428-815, can b e crystallized readily, These results suggest that FAS residues 428-48 7, 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 tran sacylase catalytic domain and may constitute an extended structural li nker that facilitates the functional interaction between the transacyl ase and acyl carrier protein domains.