A Subfamily
of Bacterial Ribokinases Utilizes a Hemithioacetal
for Pyridoxal Phosphate Salvage
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Abstract
Pyridoxal 5′-phosphate
(PLP) is the active vitamer of vitamin
B<sub>6</sub> and acts as an essential cofactor in many aspects of
amino acid and sugar metabolism. The virulence and survival of pathogenic
bacteria such as Mycobacterium tuberculosis depend on PLP, and deficiencies in humans have also been associated
with neurological disorders and inflammation. While PLP can be synthesized
by a de novo pathway in bacteria and plants, most higher organisms
rely on a salvage pathway that phosphorylates either pyridoxal (PL)
or its related vitamers, pyridoxine (PN) and pyridoxamine (PM). PL
kinases (PLKs) are essential for this phosphorylation step and are
thus of major importance for cellular viability. We recently identified
a pyridoxal kinase (SaPLK) as a target of the natural product antibiotic
rugulactone (Ru) in Staphylococcus aureus. Surprisingly, Ru selectively modified SaPLK not at the active site
cysteine, but on a remote cysteine residue. Based on structural and
biochemical studies, we now provide insight into an unprecedented
dual Cys charge relay network that is mandatory for PL phosphorylation.
The key component is the reactive Cys 110 residue in the lid region
that forms a hemithioactetal intermediate with the 4′-aldehyde
of PL. This hemithioacetal, in concert with the catalytic Cys 214,
increases the nucleophilicity of the PL 5′-OH group for the
inline displacement reaction with the γ-phosphate of ATP. A
closer inspection of related enzymes reveals that Cys 110 is conserved
and thus serves as a characteristic mechanistic feature for a dual-function
ribokinase subfamily herein termed CC-PLKs