Bacterial synthesis of active rat stearyl-CoA desaturase lacking the 26-residue amino-terminal amino acid sequence.
Two clones containing inserts in pBR322 that together include the entire 1074-base open reading frame coding for the 358 amino acids of rat liver stearyl-CoA desaturase have been used to construct expression vectors for residues 3-358 and 27-358 fused to the first 6 residues of beta-galactosidase and several amino acids of the multiple cloning site of pUC8. Growth of transformed Escherichia coli under conditions for suppression of the lac promoter, followed by subsequent induction of these cultures results in the synthesis of higher levels of desaturase proteins than those found in induced rat liver. The proteins are almost completely associated with the membrane fraction of cell homogenates. Posttranslational iron insertion into the apoproteins, either in vitro with membrane preparations or by iron addition during induction, results in the formation of active holoenzyme which can be reconstituted with NADH cytochrome b5 reductase and cytochrome b5 to form an active stearyl-CoA desaturase system. The deletion of the first 26 amino-terminal amino acid residues does not affect either enzyme activity or membrane binding. Therefore, the unusual sequence of 11 residues containing 10 amino acids with hydroxyl groups plays no apparent significant role in either protein insertion into membranes or iron chelation. Since the protein product for residues 3-358 is processed even further to delete the initial 33 amino-terminal residues, the limiting polypeptide primary structure required for an active membrane-bound catalyst is even smaller than this initial deletion mutation indicates.
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000378:metabolism; D015786:Cytochromes b5; D004926:Escherichia coli / Q000201:enzymology*; D044943:Fatty Acid Desaturases / Q000096:biosynthesis*; D007163:Immunosorbent Techniques; D007501:Iron / Q000378:metabolism; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D051381:Rats; D013230:Stearoyl-CoA Desaturase / Q000032:analysis / Q000096:biosynthesis*
D003573:Cytochrome b Group; D015786:Cytochromes b5; D007501:Iron; D044943:Fatty Acid Desaturases; D013230:Stearoyl-CoA Desaturase