title: The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: studies on the mechanisms of the reactions catalyzed by IspG and IspH protein.
abstract: Earlier in vivo studies have shown that the sequential action of the IspG and IspH proteins is essential for the reductive transformation of 2C-methyl-d-erythritol 2,4-cyclodiphosphate into dimethylallyl diphosphate and isopentenyl diphosphate via 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. A recombinant fusion protein comprising maltose binding protein and IspG protein domains was purified from a recombinant Escherichia coli strain. The purified protein failed to transform 2C-methyl-d-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate, but catalytic activity could be restored by the addition of crude cell extract from an ispG-deficient E. coli mutant. This indicates that auxiliary proteins are required, probably as shuttles for redox equivalents. On activation by photoreduced 10-methyl-5-deaza-isoalloxazine, the recombinant protein catalyzed the formation of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate from 2C-methyl-d-erythritol 2,4-cyclodiphosphate at a rate of 1 nmol x min(-1) x mg(-1). Similarly, activation by photoreduced 10-methyl-5-deaza-isoalloxazine enabled purified IspH protein to catalyze the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into a 6:1 mixture of isopentenyl diphosphate and dimethylallyl diphosphate at a rate of 0.4 micromol x min(-1) x mg(-1). IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of 3 nmol x min(-1) x mg(-1). The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions.
authors.affiliation: Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany. felix.rohdich@ch.tum.de
authors.forename: Felix
authors.initials: F
authors.lastname: Rohdich
authors.forename: Ferdinand
authors.initials: F
authors.lastname: Zepeck
authors.forename: Petra
authors.initials: P
authors.lastname: Adam
authors.forename: Stefan
authors.initials: S
authors.lastname: Hecht
authors.forename: Johannes
authors.initials: J
authors.lastname: Kaiser
authors.forename: Ralf
authors.initials: R
authors.lastname: Laupitz
authors.forename: Tobias
authors.initials: T
authors.lastname: Gräwert
authors.forename: Sabine
authors.initials: S
authors.lastname: Amslinger
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Eisenreich
authors.forename: Adelbert
authors.initials: A
authors.lastname: Bacher
authors.forename: Duilio
authors.initials: D
authors.lastname: Arigoni
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D015153:Blotting, Western; D002384:Catalysis; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D019906:Nuclear Magnetic Resonance, Biomolecular; D010088:Oxidoreductases / Q000378:metabolism*; D011106:Polyisoprenyl Phosphates / Q000096:biosynthesis*; D014996:Xylulose / Q000031:analogs & derivatives* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011106:Polyisoprenyl Phosphates; D014996:Xylulose; C029724:1-deoxy-2-pentulose; D010088:Oxidoreductases; C109866:ispH protein, E coli