Department of Applied Biotechnology, Budapest University of Technology and Economics, Budapest, Hungary.
Servier Research Institute of Medicinal Chemistry, Budapest, Hungary.
Appl Microbiol Biotechnol. 2024 Dec;108(1):101. doi: 10.1007/s00253-023-12871-z. Epub 2024 Jan 13.
Enzymatic processes play an increasing role in synthetic organic chemistry which requires the access to a broad and diverse set of enzymes. Metagenome mining is a valuable and efficient way to discover novel enzymes with unique properties for biotechnological applications. Here, we report the discovery and biocatalytic characterization of six novel metagenomic opine dehydrogenases from a hot spring environment (mODHs) (EC 1.5.1.X). These enzymes catalyze the asymmetric reductive amination between an amino acid and a keto acid resulting in opines which have defined biochemical roles and represent promising building blocks for pharmaceutical applications. The newly identified enzymes exhibit unique substrate specificity and higher thermostability compared to known examples. The feature that they preferably utilize negatively charged polar amino acids is so far unprecedented for opine dehydrogenases. We have identified two spatially correlated positions in their active sites that govern this substrate specificity and demonstrated a switch of substrate preference by site-directed mutagenesis. While they still suffer from a relatively narrow substrate scope, their enhanced thermostability and the orthogonality of their substrate preference make them a valuable addition to the toolbox of enzymes for reductive aminations. Importantly, enzymatic reductive aminations with highly polar amines are very rare in the literature. Thus, the preparative-scale enzymatic production, purification, and characterization of three highly functionalized chiral secondary amines lend a special significance to our work in filling this gap. KEY POINTS: • Six new opine dehydrogenases have been discovered from a hot spring metagenome • The newly identified enzymes display a unique substrate scope • Substrate specificity is governed by two correlated active-site residues.
酶促过程在合成有机化学中扮演着越来越重要的角色，这需要获得广泛而多样的酶。宏基因组挖掘是发现具有独特性质的新型酶的一种有价值且有效的方法，这些酶可用于生物技术应用。在这里，我们报道了从温泉环境中发现的六种新型宏基因组阿朴啡脱氢酶（mODH）（EC 1.5.1.X）及其生物催化特性。这些酶催化氨基酸和酮酸之间的不对称还原胺化反应，生成具有明确生化作用的阿朴啡，它们是药物应用有前途的构建模块。新鉴定的酶与已知的酶相比具有独特的底物特异性和更高的热稳定性。到目前为止，它们优先利用带负电荷的极性氨基酸的特性在阿朴啡脱氢酶中是前所未有的。我们在它们的活性位点中鉴定出两个空间相关的位置，这些位置控制着这种底物特异性，并通过定点突变证明了底物偏好的转变。虽然它们仍然存在相对较窄的底物范围，但它们增强的热稳定性和底物偏好的正交性使它们成为还原胺化酶工具包的有价值的补充。重要的是，在文献中，用高极性胺进行酶促还原胺化非常罕见。因此，三种高功能化手性仲胺的酶法制备规模生产、纯化和特性分析使我们的工作在填补这一空白方面具有特殊意义。关键点： • 从温泉宏基因组中发现了六种新的阿朴啡脱氢酶 • 新鉴定的酶具有独特的底物范围 • 底物特异性由两个相关的活性位点残基控制。
