A revised mechanism for (p)ppGpp synthesis by Rel proteins: The critical role of the 29-OH of GTP.

Bacterial Rel proteins synthesize hyperphosphorylated guanosine nucleotides, denoted as (p)ppGpp, which by inhibiting
energy requiring molecular pathways help bacteria to overcome
the depletion of nutrients in its surroundings. (p)ppGpp synthesis by Rel involves transferring a pyrophosphate from ATP to
the oxygen of 39-OH of GTP/GDP. Initially, a conserved glutamate at the active site was believed to generate the nucleophile
necessary to accomplish the reaction. Later this role was alluded
to a Mg21 ion. However, no study has unequivocally established
a catalytic mechanism for (p)ppGpp synthesis. Here we present
a revised mechanism, wherein for the first time we explore a
role for 29-OH of GTP and show how it is important in generating the nucleophile. Through a careful comparison of substratebound structures of Rel, we illustrate that the active site does
not discriminate GTP from dGTP, for a substrate. Using biochemical studies, we demonstrate that both GTP and dGTP
bind to Rel, but only GTP (but not dGTP) can form the product.
Reactions performed using GTP analogs substituted with different chemical moieties at the 29 position suggest a clear role for
29-OH in catalysis by providing an indispensable hydrogen
bond; preliminary computational analysis further supports this
view. This study elucidating a catalytic role for 29-OH of GTP in
(p)ppGpp synthesis allows us to propose different mechanistic
possibilities by which it generates the nucleophile for the synthesis reaction. This study underscores the selection of ribose
nucleotides as second messengers and finds its roots in the old
RNA world hypothesis.