Specificity and Reactivity of Mycobacterium tuberculosis Serine/Threonine Kinases PknG and PknB

Mycobacterium tuberculosis (Mtb),
the causative agent of Tuberculosis, has 11 eukaryotic-like serine/threonine
protein kinases, which play essential roles in cell growth, signal
transduction, and pathogenesis. Protein kinase G (PknG) regulates
the carbon and nitrogen metabolism by phosphorylation of the glycogen
accumulation regulator (GarA) protein at Thr21. Protein kinase B (PknB)
is involved in cell wall synthesis and cell shape, as well as phosphorylates
GarA but at Thr22. While PknG seems to be constitutively activated
and recognition of GarA requires phosphorylation in its unstructured
tail, PknB activation is triggered by phosphorylation of its activation
loop, which allows binding of the forkhead-associated domain of GarA.
In the present work, we used molecular dynamics and quantum-mechanics/molecular
mechanics simulations of the catalytically competent complex and kinase
activity assays to understand PknG/PknB specificity and reactivity
toward GarA. Two hydrophobic residues in GarA, Val24 and Phe25, seem
essential for PknG binding and allow specificity for Thr21 phosphorylation.
On the other hand, phosphorylated residues in PknB bind Arg26 in GarA
and regulate its specificity for Thr22. We also provide a detailed
analysis of the free energy profile for the phospho-transfer reaction
and show why PknG has a constitutively active conformation not requiring
priming phosphorylation in contrast to PknB. Our results provide new
insights into these two key enzymes relevant for Mtb and the mechanisms
of serine/threonine phosphorylation in bacteria.