Functional characterization of GcpE, an essential enzyme of the non-mevalonate pathway of isoprenoid biosynthesis.
The gcpE gene product controls one of the terminal steps of isoprenoid biosynthesis via the mevalonate independent 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. This pathway is utilized by a variety of eubacteria, the plastids of algae and higher plants, and the plastid-like organelle of malaria parasites. Recombinant GcpE protein from the hyperthermophilic bacterium Thermus thermophilus was produced in Escherichia coli and purified under dioxygen-free conditions. The protein was enzymatically active in converting 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the presence of dithionite as reductant. The maximal specific activity was 0.6 micromol x min(-1) x mg(-1) at pH 7.5 and 55 degrees C. The kcat value was 0.4 s(-1) and the K(m) value for HMBPP 0.42 mM.
Kollas
Ann-Kristin
AK
Biochemisches Institut, Justus-Liebig-Universität Giessen, Friedrichstrasse 24, 35392, Giessen, Germany.
Duin
Evert C
EC
Eberl
Matthias
M
Altincicek
Boran
B
Hintz
Martin
M
Reichenberg
Armin
A
Henschker
Dajana
D
Henne
Anke
A
Steinbrecher
Irina
I
Ostrovsky
Dmitry N
DN
Hedderich
Reiner
R
Beck
Ewald
E
Jomaa
Hassan
H
Wiesner
Jochen
J
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008027:Light; D008213:Lymphocyte Activation; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010755:Organophosphates / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D018087:Plastids / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000096:biosynthesis*; D011743:Pyrimidines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013601:T-Lymphocytes / Q000378:metabolism; D016963:Thermus thermophilus / Q000378:metabolism; D014466:Ultraviolet Rays
C467061:4-hydroxy-3-methyl-2-butenyl diphosphate; D001426:Bacterial Proteins; D004798:Enzymes; D010755:Organophosphates; D011106:Polyisoprenyl Phosphates; D011743:Pyrimidines; D011994:Recombinant Proteins; C421242:hydroxymethylbutenyl 4-diphosphate synthase; C078327:2-methyl-5,6-cyclopentapyrimidine; D010100:Oxygen