Conformational Dynamics Contribute to Substrate Selectivity and Catalysis in Human Kynureninase

Abstract

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.

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

“…Beyond epitope mapping, HDX-MS continues to be utilized for studying protein–protein interactions in complex protein systems and revealing mechanisms of activation in a range of systems including: clotting factors, 320 , 327 − 329 metabolic enzymes, 67 , 298 , 330 − 338 heat shock proteins 317 , 339 , 340 and other chaperones, 341 − 348 signaling complexes, 349 − 360 and complexes exclusively residing in membranes. 361 HDX-MS has also been extensively used to study interactions between proteins with nucleic acids, including activation of transcription factors.…”

“…Beyond epitope mapping, HDX-MS continues to be utilized for studying protein–protein interactions in complex protein systems and revealing mechanisms of activation in a range of systems including: clotting factors, ,− metabolic enzymes, ,,− heat shock proteins ,, and other chaperones, − signaling complexes, − and complexes exclusively residing in membranes . HDX-MS has also been extensively used to study interactions between proteins with nucleic acids, including activation of transcription factors. − Other notable applications are the characterization of ligands with weak affinity such as metals ,− or carbohydrates. , For proteins with multiple distinct ligand binding sites, it has been shown that by performing a concentration series it is possible to characterize individual interactions. , The strength of HDX-MS to provide detailed insight into such a wide range of interactions continues to drive its use to study increasingly complex biological systems.…”

Engineering of the Recombinant Expression and PEGylation Efficiency of the Therapeutic Enzyme Human Thymidine Phosphorylase

“…Taken together, our data demonstrate that by rational selection of suitable arginine and lysine residues for mutagenesis, PEGylation efficiency and consistency can be significantly improved while preserving the catalytic activity post-PEGylation. Our experimentally-validated rational PEGylation design may be applicable and benefit other protein and enzyme therapeutic molecules including asparaginase (Karamitros and Konrad, 2016;Meneguetti et al, 2019), cysteinase (Cramer et al, 2017) and kynureninase (Triplett et al, 2018;Karamitros et al, 2020).…”

Discovery of Highly Active Kynureninases for Cancer Immunotherapy through Protein Language Model

“…Determination of kinetic parameters. The steady-state specific activity assays were carried out, as reported previously 5,12 . In short, we mixed 20 µL of enzyme in DPBS buffer with 80 µL of L-KYN (Sigma-Aldrich) at 37 °C.…”

“…All four putative KYNases we selected show comparable or even substantially higher kinetic parameters than Pf-K 5,12 (Fig. 2b, Supplementary Fig.…”