Overproduction and one-step purification of Escherichia coli 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase and oxygen transfer studies during catalysis using isotopic-shifted heteronuclear NMR.
The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase catalyzes the condensation of D-arabinose 5-phosphate with phosphoenolpyruvate to give the unique 8-carbon acidic sugar 3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO 8-P) found only in Gram-negative bacteria and required for lipid A maturation and cellular growth. The Escherichia coli gene kdsA that encodes KDO 8-P synthase has been amplified by polymerase chain reaction methodologies and subcloned into the expression vector, pT7-7. A simple one-step purification yields 200 mg of homogeneous KDO 8-P synthase per liter of cell culture. [2-13C,18O]Phosphoenolpyruvate (PEP) was prepared by first, exchange of [2-13C]-3-bromopyruvate with 2H2 18O followed by reaction of the labeled bromopyruvate with trimethylphosphite. The fate of the enolic oxygen in this multilabeled PEP, during the course of the KDO 8-P synthase-catalyzed reaction with D-arabinose 5-phosphate, was monitored by 13C and 31P NMR spectroscopy. The inorganic phosphate formed during the reaction was further analyzed via mass spectral analysis of its trimethyl ester derivative. The 13C NMR spectrum of an incubation mixture of [2-13C]PEP and D-arabinose 5-phosphate in 2H2 18O in the presence of KDO 8-P synthase was also recorded. [2-13C]KDO 8-P was utilized to determine the extent of nonenzymatic incorporation of 18O into the C-2 position of KDO 8-P. The results indicate that the enolic oxygen of the PEP is recovered with the inorganic phosphate, and the C-2 oxygen of KDO 8-P originates from the solvent, H2O.
Dotson
G D
GD
College of Pharmacy, University of Michigan, Ann Arbor 48109-1065, USA.
Dua
R K
RK
Clemens
J C
JC
Wooten
E W
EW
Woodard
R W
RW
D000446:Aldehyde-Lyases / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism*
C020011:2-dehydro-3-deoxyphosphooctonate aldolase; D000446:Aldehyde-Lyases; D010100:Oxygen