Site-directed mutagenesis of the spinach acyl carrier protein-I prosthetic group attachment site.
Site-directed mutagenesis was used to change the phosphopantetheine attachment site (Ser38) of spinach acyl carrier protein I (ACP-I) from a serine to a threonine or cysteine residue. 1. Although the native ACP-I is fully phosphopantethenylated when expressed in Escherichia coli, the TH-ACP-I and CY-ACP-I mutants were found to be completely devoid of the phosphopantetheine group. Therefore, the E. coli holoACP synthase requires serine for in vivo phosphopantetheine addition to spinach ACP-I. 2. Spinach holoACP synthase was completely inactive in vitro with either the TH-ACP-I or CY-ACP-I mutants. In addition, TH-ACP-I and CY-ACP-I were strong inhibitors of spinach holoACP synthase. 3. The mutant ACPs were weak or ineffective as inhibitors of spinach fatty acid synthesis and spinach oleoyl-ACP hydrolase. 4. Compared to holoACP-I, the mutant apoACP-I analogs had: (a) altered mobility in SDS and native gel electrophoresis, (b) altered binding to anti-(spinach ACP-I) antibodies and (c) altered isoelectric points. The combined physical, immunological and enzyme inhibition data indicate that attachment of the phosphopantheine prosthetic group alters ACP conformation.
Jaworski
J G
JG
Department of Botany and Plant Pathology, Michigan State University.
Post-Beittenmiller
M A
MA
Ohlrogge
J B
JB
D000213:Acyl Carrier Protein / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D003429:Cross Reactions; D004586:Electrophoresis; D004926:Escherichia coli / Q000378:metabolism; D007525:Isoelectric Focusing; D009154:Mutation; D010204:Pantetheine / Q000031:analogs & derivatives; D010770:Phosphotransferases / Q000037:antagonists & inhibitors; D010944:Plants / Q000032:analysis*; D011487:Protein Conformation; D017855:Transferases (Other Substituted Phosphate Groups)
D000213:Acyl Carrier Protein; D010204:Pantetheine; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase; C003129:4'-phosphopantetheine