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21-04-07 @ 19:53
Chuxray  
Crystallographic analysis of initial modes of liga
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The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS)
catalyses the condensation of arabinose 5-phosphate (A5P) and phosphoenol pyruvate
(PEP) to obtain 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P). We have
elucidated the initial modes of ligand binding in KDO8PS binary complexes by X-ray
crystallographic analysis. The structure of the ligand-free apo-KDO8PS visualizes for the
first time the role of His202 as an active-site gate. Five additional crystal structures were
obtained by incubation of the apo-KDO8PS crystals with PEP, the E and Z isomers of 3-
fluoro-PEP, a catalytically inactive 1-deoxy analogue of arabinose 5-phosphate (1dA5P)
and the product KDO8P. These structures visualize the manner by which each ligand
binds to the enzyme prior to the initiation of catalysis. The KDO8PS active site resembles
an irregular funnel with positive electrostatic potential situated at the bottom of the PEP
binding sub-site, which is the primary attractive force towards negatively charged
phosphate moieties of all ligands. Actual positioning of the ligands is however
determined by residues higher up within the funnel, especially His202. The structures of
the fluoro-PEP isomers explain for the first time the ~27-fold selectivity of the enzyme
towards one geometric isomer of its fluorinated substrate relative to the other.
Examination of the crystal structures of KDO8PS with the KDO8P or 1dA5P, show these
ligands bound to the enzyme in the PEP binding sub-site, and not as expected to the A5P
sub-site. Taken together, the structures presented here strengthen earlier evidence that this
enzyme predominately functions through positional catalysis, maps out the roles of active
site residues and provides evidence that explains the total lack of catalytic reversibility.
Crystal structures of KDO8P synthase from E. coli
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