Biosynthesis of terpenoids. 2C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) from Plasmodium falciparum.
The putative catalytic domain of an open reading frame from Plasmodium falciparum with similarity to the ispF gene of Escherichia coli specifying 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase was expressed in a recombinant E. coli strain. The recombinant protein was purified to homogeneity and was found to catalyze the formation of 2C-methyl-D-erythritol 2,4-cyclodiphosphate from 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate at a rate of 4.3 micromol x mg(-1) x min(-1). At lower rates, the recombinant protein catalyzes the formation of 2-phospho-2C-methyl-D-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate and the formation of 2C-methyl-D-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-D-erythritol. Divalent metal ions such as magnesium or manganese are required for catalytic activity. The enzyme has a pH optimum at pH 7.0. Recombinant expression of the full-length open reading frame afforded insoluble protein that could not be folded in vitro. The enzyme is a potential target for antimalarial drugs directed at the nonmevalonate pathway of isoprenoid biosynthesis.
Rohdich
F
F
Institut für Organische Chemie und Biochemie, Lehrstuhl III, Technische Universität München, Garching, Germany. felix.rohdich@ch.tum.de
Eisenreich
W
W
Wungsintaweekul
J
J
Hecht
S
S
Schuhr
C A
CA
Bacher
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008969:Molecular Sequence Data; D015394:Molecular Structure; D019761:Phosphorus-Oxygen Lyases; D010957:Plasmids; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D013729:Terpenes / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D013729:Terpenes; D019761:Phosphorus-Oxygen Lyases; C400181:ISPF protein, E coli