Department of Chemical Engineering, University of Texas at Austin, Austin, TX 78712.
Department of Chemistry and Biochemistry, University of Texas at Dallas, Richardson, TX 75080.
Proc Natl Acad Sci U S A. 2022 Jun 7;119(23):e2118979119. doi: 10.1073/pnas.2118979119. Epub 2022 Jun 3.
Dynamic motions of enzymes occurring on a broad range of timescales play a pivotal role in all steps of the reaction pathway, including substrate binding, catalysis, and product release. However, it is unknown whether structural information related to conformational flexibility can be exploited for the directed evolution of enzymes with higher catalytic activity. Here, we show that mutagenesis of residues exclusively located at flexible regions distal to the active site of Homo sapiens kynureninase (HsKYNase) resulted in the isolation of a variant (BF-HsKYNase) in which the rate of the chemical step toward kynurenine was increased by 45-fold. Mechanistic pre–steady-state kinetic analysis of the wild type and the evolved enzyme shed light on the underlying effects of distal mutations (>10 Å from the active site) on the rate-limiting step of the catalytic cycle. Hydrogen-deuterium exchange coupled to mass spectrometry and molecular dynamics simulations revealed that the amino acid substitutions in BF-HsKYNase allosterically affect the flexibility of the pyridoxal-5′-phosphate (PLP) binding pocket, thereby impacting the rate of chemistry, presumably by altering the conformational ensemble and sampling states more favorable to the catalyzed reaction.
酶在广泛的时间尺度上发生的动态运动在反应途径的所有步骤中都起着关键作用，包括底物结合、催化和产物释放。然而，目前尚不清楚与构象灵活性相关的结构信息是否可以用于定向进化具有更高催化活性的酶。在这里，我们表明，仅对位于人源犬尿氨酸酶（HsKYNase）活性位点远端的柔性区域的残基进行诱变，就可以分离出一种变体（BF-HsKYNase），其中犬尿氨酸形成反应的化学步骤速率提高了 45 倍。对野生型和进化酶的预稳态动力学分析揭示了活性位点以外的远端突变（>10 Å）对催化循环限速步骤的潜在影响。与质谱和分子动力学模拟相结合的氢氘交换揭示了 BF-HsKYNase 中的氨基酸取代通过变构影响 PLP 结合口袋的柔性，从而影响化学速率，可能是通过改变更有利于催化反应的构象集合和采样状态。
