Purification and enzymatic characterization of the geranylgeranyl pyrophosphate synthase from Erwinia uredovora after expression in Escherichia coli.
Geranylgeranyl pyrophosphate (GGPP) synthase from Erwinia uredovora was overexpressed in Escherichia coli and purified to homogeneity from solubilized inclusion bodies. In this protein the first 13 N-terminal amino acids were replaced by 16 other amino acids resulting from the cloning vector pUC18. Nevertheless, the enzyme showed activity after purification which could be stimulated sixfold by appropriate activation conditions. The homogeneous enzyme was used to study substrate and product specificity as well as to determine Km values for isopentenyl pyrophosphate, dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), and farnesyl pyrophosphate (FPP). Reaction rates and Km values indicate that FPP and GPP are the genuine allylic substrates for GGPP synthase, but not DMAPP. Independent of the allylic substrate employed, GGPP was the only reaction product of the enzymatic reaction.
Wiedemann
M
M
Lehrstuhl für Physiologie und Biochemie der Pflanzen, Universität Konstanz, Germany.
Misawa
N
N
Sandmann
G
G
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004885:Erwinia / Q000201:enzymology*; D004926:Escherichia coli / Q000254:growth & development; D051231:Farnesyltranstransferase; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014166:Transferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D011994:Recombinant Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051231:Farnesyltranstransferase