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.

中文翻译：

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构象动力学有助于人类Kynureninase中的底物选择性和催化作用。

核尿激酶（KYNases）是一种酶，通过降解中间犬尿氨酸和3'-羟基犬尿氨酸代谢产物（分别为KYN和OH-KYN）在色氨酸分解代谢中起关键作用。细菌KYNase对KYN的催化效率高，对OH-KYN的活性中等，而动物KYNase对OH-KYN的选择性高，对较小的KYN底物仅表现出最小的活性。这些差异反映了各个王国中KYN和OH-KYN利用的不同途径。我们检查了智人和荧光假单胞菌KYNases（分别为HsKYNase和PfKYNase）使用稳态前和氢氘交换质谱法（HDX-MS）。我们发现，HsKYNase的活性关键取决于与OH-KYN羟基形成氢键，以稳定整个活性位点并允许生产性底物更新。对于优选的OH-KYN底物，在底物结合位点和PLP辅因子周围的区域观察到稳定。使用非优选的KYN底物，稳定性降低，显示与活性直接相关。这种相关性对于PfKYNase成立。然而，在底物结合位点仅有适度的稳定作用，这表明仅通过空间位阻即可实现底物识别。我们推测，真核KYNases使用动态迁移率作为底物特异性的机制，将OH-KYN提交给烟酰胺合成，并避免了KYN徒劳的水解。这些发现对于癌症免疫疗法临床应用所需的具有高KYN活性的HsKynase的工程改造具有重要意义。我们的研究表明，具有保守活性位点的同源酶如何利用动力学来区分两种高度相似的底物。