Disruption of
an Active Site Network Leads to Activation
of C2α-Lactylthiamin Diphosphate on the Antibacterial Target
1‑Deoxy‑d‑xylulose-5-phosphate Synthase
posted on 2024-02-23, 16:04authored byEucolona
M. Toci, Steven L. Austin, Ananya Majumdar, H. Lee Woodcock, Caren L. Freel Meyers
The bacterial metabolic enzyme 1-deoxy-d-xylulose-5-phosphate
synthase (DXPS) catalyzes the thiamin diphosphate (ThDP)-dependent
formation of DXP from pyruvate and d-glyceraldehyde-3-phosphate
(d-GAP). DXP is an essential bacteria-specific metabolite
that feeds into the biosynthesis of isoprenoids, pyridoxal phosphate
(PLP), and ThDP. DXPS catalyzes the activation of pyruvate to give
the C2α-lactylThDP (LThDP) adduct that is long-lived on DXPS
in a closed state in the absence of the cosubstrate. Binding of d-GAP shifts the DXPS-LThDP complex to an open state which coincides
with LThDP decarboxylation. This gated mechanism distinguishes DXPS
in ThDP enzymology. How LThDP persists on DXPS in the absence of cosubstrate,
while other pyruvate decarboxylases readily activate LThDP for decarboxylation,
is a long-standing question in the field. We propose that an active
site network functions to prevent LThDP activation on DXPS until the
cosubstrate binds. Binding of d-GAP coincides with a conformational
shift and disrupts the network causing changes in the active site
that promote LThDP activation. Here, we show that the substitution
of putative network residues, as well as nearby residues believed
to contribute to network charge distribution, predictably affects
LThDP reactivity. Substitutions predicted to disrupt the network have
the effect to activate LThDP for decarboxylation, resulting in CO2 and acetate production. In contrast, a substitution predicted
to strengthen the network fails to activate LThDP and has the effect
to shift DXPS toward the closed state. Network-disrupting substitutions
near the carboxylate of LThDP also have a pronounced effect to shift
DXPS to an open state. These results offer initial insights to explain
the long-lived LThDP intermediate and its activation through disruption
of an active site network, which is unique to DXPS. These findings
have important implications for DXPS function in bacteria and its
development as an antibacterial target.