Role of the Occluded Conformation in Bacterial Dihydrofolate Reductases
2014
Dihydrofolate reductase (DHFR) from Escherichia
coli (EcDHFR) adopts two major conformations, closed
and occluded, and movement between these two conformations
is important for progression through the catalytic cycle.
DHFR from the cold-adapted organism Moritella profunda
(MpDHFR) on the other hand is unable to form the two
hydrogen bonds that stabilize the occluded conformation in
EcDHFR and so remains in a closed conformation during
catalysis. EcDHFR-S148P and MpDHFR-P150S were examined
to explore the influence of the occluded conformation on
catalysis by DHFR. Destabilization of the occluded conformation did not affect hydride transfer but altered the affinity for the
oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) and changed the rate-determining step of the catalytic
cycle for EcDHFR-S148P. Even in the absence of an occluded conformation, MpDHFR follows a kinetic pathway similar to that
of EcDHFR with product release being the rate-limiting step in the steady state at pH 7, suggesting that MpDHFR uses a
different strategy to modify its affinity for NADP+. DHFRs from many organisms lack a hydrogen bond donor in the appropriate
position and hence most likely do not form an occluded conformation. The link between conformational cycling between closed
and occluded forms and progression through the catalytic cycle is specific to EcDHFR and not a general characteristic of
prokaryotic DHFR catalysis.
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