Isoprenoid biosynthesis in plants - 2C-methyl-D-erythritol-4-phosphate synthase (IspC protein) of Arabidopsis thaliana.
The ispC gene of Arabidopsis thaliana was expressed in pseudomature form without the putative plastid-targeting sequence in a recombinant Escherichia coli strain. The recombinant protein was purified by affinity chromatography and was shown to catalyze the formation of 2C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate at a rate of 5.6 micromol x min(-1) x mg(-1) (k(cat) 4.4 s(-1)). The Michaelis constants for 1-deoxy-D-xylulose 5-phosphate and the cosubstrate NADPH are 132 and 30 microm, respectively. The enzyme has an absolute requirement for divalent metal ions, preferably Mn2+ and Mg2+, and is inhibited by fosmidomycin with a Ki of 85 nm. The pH optimum is 8.0. NADH can substitute for NADPH, albeit at a low rate (14% as compared to NADPH). The enzyme catalyzes the reverse reaction at a rate of 2.1 micromol x min(-1) x mg(-1).
Rohdich
Felix
F
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Garching, Germany. felix.rohdich@ch.tum.de
Lauw
Susan
S
Kaiser
Johannes
J
Feicht
Richard
R
Köhler
Peter
P
Bacher
Adelbert
A
Eisenreich
Wolfgang
W
D019747:Aldose-Ketose Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D005578:Fosfomycin / Q000031:analogs & derivatives / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D013729:Terpenes / Q000378:metabolism*
D029681:Arabidopsis Proteins; D009097:Multienzyme Complexes; D013729:Terpenes; D005578:Fosfomycin; D009249:NADP; C024640:fosmidomycin; D010088:Oxidoreductases; C114231:1-deoxy-D-xylulose 5-phosphate reductoisomerase; D019747:Aldose-Ketose Isomerases; D008274:Magnesium