Analysis of Enzyme Dynamics in Kynureninase Through Hydrogendeuterium Exchange Mass Spectrometry



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Roughly 38.5% of people will be diagnosed with cancer in their lifetime1. Elevated levels of the small molecule, kynurenine, is a feature of the microtumor environment in 58% of all cancers2. Kynurenine promotes immunosuppression and is compounded via an inflammation-directed positive feedback loop. Kynureninase is an enzyme capable of breaking down kynurenine to ultimately restore immune response and dismantle the positive feedback loop. Unfortunately, the human version of this enzyme has low preference for kynurenine, with high selectivity toward the similar 3’-hydroxylated kynurenine. In this dissertation, human kynureninase was compared with a kynurenine-selective bacterial kynureninase homolog. Bacterial kynureninase was compared against the human ortholog using kinetic assays, Molecular Dynamics, and hydrogen-deuterium exchange mass spectrometry. With the knowledge that both enzymes can break down either substrate, we discovered that substrate preference directs ratelimiting steps using pre-steady-state kinetics. Molecular Dynamics complemented experimental data from hydrogen-deuterium exchange mass spectrometry to describe key regions and even residues critical for enzyme catalysis. We identified two critical features required for efficient catalysis. The first is human motif containing residues 305-338. This region contains rigid secondary structure in the human enzyme yet appears flexible in the bacterial homolog which lacks secondary structure. Secondly, kynureninases have a PLP cofactor that coordinates several residues in the active site. One region, an -helix cap, associated with human residues 136-137 contains key helix-helix contacts with residues adjacent to F165. These two regions are what bottlenecks the efficiency of the human enzyme towards kynurenine. Mutated KYNase, which show improved efficiency toward kynurenine demonstrate increased flexibility of the helix-helix contact. Protein engineering efforts led to two varieties of mutated human kynureninase. The first variety (B-Factor kynureninases) was a result of distal mutations resulting in up to an 11-fold increase in kynurenine efficiency. The second variety (bacterialized kynureninases) resulted from directed evolution, generating two versions of human kynureninase that were either 114- or 275-fold more efficient towards kynurenine than the human wildtype. Efficiency was measured in terms of kcat/KM. We discovered that flexibility in the 305-338 was maintained throughout the improved variants. As efficiency towards kynurenine increased, stabilization occurred across all other loops that transversed the active site. Ultimately, we have described wildtype and engineered kynureninases in detail to best understand how catalytic events within enzymes, specifically kynureninase, are sensitive to a wide variety of perturbations which lead to a dramatic increase in efficiency.



Hydrogen, Deuterium, Mass spectrometry, Molecular dynamics, Kynurenine, Enzymes, Protein engineering