Biosynthesis of isoprenoids. purification and properties of IspG protein from Escherichia coli.
[Structure: see text]. The IspG protein is known to catalyze the transformation of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate in the nonmevalonate pathway of isoprenoid biosynthesis. We have found that the apparent IspG activity in the cell extracts of recombinant Escherichia coli cells as observed by a radiochemical assay can be enhanced severalfold by coexpression of the isc operon which is involved in the assembly of iron-sulfur clusters. The recombinant protein was isolated by affinity chromatography under anaerobic conditions. With a mixture of flavodoxin, flavodoxin reductase, and NADPH as the reducing agent, stringent assay methods based on photometry or on 13C NMR detection of multiply 13C-labeled substrate/product ratios afforded catalytic activities greater than 60 nmol mg(-1) min(-1) for the protein "as isolated" (i.e., without reconstitution of any kind). Lower apparent activities were found using photoreduced deazaflavin as an artifactual electron donor, whereas dithionite was unable to serve as an artificial electron donor. The apparent Michaelis constant for 2-C-methyl-D-erythritol 2,4-cyclodiphosphate was 700 microM. The enzyme was inactivated by EDTA and could be reactivated by Mn2+. The pH optimum was at 9.0. The protein contained 2.4 iron ions and 4.4 sulfide ions per subunit. The replacement of any of the three conserved cysteine residues afforded mutant proteins which were devoid of catalytic activity and contained less than 6% of Fe2+ and less than 23% of S2- as compared to the wild-type protein. Sequence comparison indicates that putative IspG proteins of plants, the apicomplexan protozoan Plasmodium falciparum, and bacteria from the Bacteroidetes/Chlorobi group contain an insert of about 170-320 amino acid residues as compared with eubacterial enzymes.
Zepeck
Ferdinand
F
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany.
Gräwert
Tobias
T
Kaiser
Johannes
J
Schramek
Nicholas
N
Eisenreich
Wolfgang
W
Bacher
Adelbert
A
Rohdich
Felix
F
D000595:Amino Acid Sequence; D002384:Catalysis; D002846:Chromatography, Affinity; D004896:Erythritol / Q000031:analogs & derivatives / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005418:Flavodoxin / Q000378:metabolism; D007501:Iron / Q000737:chemistry; D008798:Mevalonic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013440:Sulfides / Q000737:chemistry; D013729:Terpenes / Q000378:metabolism*
D029968:Escherichia coli Proteins; D005418:Flavodoxin; D011994:Recombinant Proteins; D013440:Sulfides; D013729:Terpenes; C443879:methyl-D-erythritol 2,4-cyclodiphosphate; D009249:NADP; D007501:Iron; D009247:NADH, NADPH Oxidoreductases; C116041:flavodoxin NADPH oxidoreductase; D004896:Erythritol; D008798:Mevalonic Acid