Conformational Dynamics Contribute to Substrate Selectivity and Catalysis in Human Kynureninase

Kynureninases
(KYNases) are enzymes that play a key role in tryptophan
catabolism through the degradation of intermediate kynurenine and
3′-hydroxy-kynurenine metabolites (KYN and OH-KYN, respectively).
Bacterial KYNases exhibit high catalytic efficiency toward KYN and
moderate activity toward OH-KYN, whereas animal KYNases are highly
selective for OH-KYN, exhibiting only minimal activity toward the
smaller KYN substrate. These differences reflect divergent pathways
for KYN and OH-KYN utilization in the respective kingdoms. We examined
the Homo sapiens and Pseudomonas fluorescens KYNases (HsKYNase and PfKYNase respectively) using pre-steady-state
and hydrogen–deuterium exchange mass spectrometry (HDX-MS)
methodologies. We discovered that the activity of HsKYNase critically
depends on formation of hydrogen bonds with the hydroxyl group of
OH-KYN to stabilize the entire active site and allow productive substrate
turnover. With the preferred OH-KYN substrate, stabilization is observed
at the substrate-binding site and the region surrounding the PLP cofactor.
With the nonpreferred KYN substrate, less stabilization occurs, revealing
a direct correlation with activity. This correlation holds true for
PfKYNases; however there is only a modest stabilization at the substrate-binding
site, suggesting that substrate discrimination is simply achieved
by steric hindrance. We speculate that eukaryotic KYNases use dynamic
mobility as a mechanism of substrate specificity to commit OH-KYN
to nicotinamide synthesis and avoid futile hydrolysis of KYN. These
findings have important ramifications for the engineering of HsKynase
with high KYN activity as required for clinical applications in cancer
immunotherapy. Our study shows how homologous enzymes with conserved
active sites can use dynamics to discriminate between two highly similar
substrates.