Institute for Biotechnology and Biochemical Engineering, Graz University of Technology, Graz, Austria.
PLoS One. 2013 Jul 23;8(7):e68932. doi: 10.1371/journal.pone.0068932. Print 2013.
(S)-Hydroxymandelate synthase (Hms) is a nonheme Fe(II) dependent dioxygenase that catalyzes the oxidation of 4-hydroxyphenylpyruvate to (S)-4-hydroxymandelate by molecular oxygen. In this work, the substrate promiscuity of Hms is characterized in order to assess its potential for the biosynthesis of chiral α-hydroxy acids. Enzyme kinetic analyses, the characterization of product spectra, quantitative structure activity relationship (QSAR) analyses and in silico docking studies are used to characterize the impact of substrate properties on particular steps of catalysis. Hms is found to accept a range of α-oxo acids, whereby the presence of an aromatic substituent is crucial for efficient substrate turnover. A hydrophobic substrate binding pocket is identified as the likely determinant of substrate specificity. Upon introduction of a steric barrier, which is suspected to obstruct the accommodation of the aromatic ring in the hydrophobic pocket during the final hydroxylation step, the racemization of product is obtained. A steady state kinetic analysis reveals that the turnover number of Hms strongly correlates with substrate hydrophobicity. The analysis of product spectra demonstrates high regioselectivity of oxygenation and a strong coupling efficiency of C-C bond cleavage and subsequent hydroxylation for the tested substrates. Based on these findings the structural basis of enantioselectivity and enzymatic activity is discussed.
(S)-海因酶（Hms）是一种非血红素铁(II)依赖性双加氧酶，能够通过分子氧将 4-羟基苯丙酮酸氧化为 (S)-4-羟基扁桃酸。在这项工作中，我们对 Hms 的底物混杂性进行了表征，以评估其在手性 α-羟基酸生物合成中的潜在应用。通过酶动力学分析、产物谱特征分析、定量构效关系（QSAR）分析和计算机对接研究，我们对底物性质对催化特定步骤的影响进行了特征描述。结果表明，Hms 可以接受一系列的 α-氧代酸，其中芳香取代基的存在对于有效的底物转化至关重要。一个疏水性的底物结合口袋被确定为底物特异性的可能决定因素。当引入一个空间位阻，这可能会阻碍芳香环在最后一步羟化过程中容纳在疏水性口袋中时，就会得到产物的外消旋化。稳态动力学分析表明，Hms 的周转率与底物疏水性强烈相关。产物谱分析表明，对于测试的底物，氧合具有高度的区域选择性，并且 C-C 键断裂和随后的羟化具有很强的耦合效率。基于这些发现，讨论了对映选择性和酶活性的结构基础。
