Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Chemical and Pharmaceutical Biology, University of Groningen, 9713 AV Groningen, The Netherlands.
Biomolecular Mechanisms, Max Planck Institute for Medical Research, 69121 Heidelberg, Germany.
Cell Chem Biol. 2021 Jun 17;28(6):876-886.e4. doi: 10.1016/j.chembiol.2021.04.010. Epub 2021 May 5.
O-Methyltransferases are ubiquitous enzymes involved in biosynthetic pathways for secondary metabolites such as bacterial antibiotics, human catecholamine neurotransmitters, and plant phenylpropanoids. While thousands of putative O-methyltransferases are found in sequence databases, few examples are functionally characterized. From a pathway engineering perspective, however, it is crucial to know the substrate and product ranges of the respective enzymes to fully exploit their catalytic power. In this study, we developed an in vitro prototyping workflow that allowed us to screen ∼30 enzymes against five substrates in 3 days with high reproducibility. We combined in vitro transcription/translation of the genes of interest with a microliter-scale enzymatic assay in 96-well plates. The substrate conversion was indirectly measured by quantifying the consumption of the S-adenosyl-L-methionine co-factor by time-resolved fluorescence resonance energy transfer rather than time-consuming product analysis by chromatography. This workflow allowed us to rapidly prototype thus far uncharacterized O-methyltransferases for future use as biocatalysts.
O-甲基转移酶是参与生物合成途径的普遍存在的酶，这些途径包括细菌抗生素、人类儿茶酚胺神经递质和植物苯丙烷类化合物等次生代谢物。虽然在序列数据库中发现了数千种假定的 O-甲基转移酶，但只有少数具有功能特征。然而，从途径工程的角度来看，了解各个酶的底物和产物范围对于充分发挥其催化能力至关重要。在这项研究中，我们开发了一种体外原型制作工作流程，该流程允许我们在 3 天内用高重复性筛选对 5 种底物具有约 30 种酶。我们将感兴趣基因的体外转录/翻译与 96 孔板中的微升规模酶测定相结合。通过时间分辨荧光共振能量转移间接测量 S-腺苷甲硫氨酸辅因子的消耗来间接测量底物转化，而不是通过色谱法进行耗时的产物分析。该工作流程使我们能够快速原型化迄今为止尚未表征的 O-甲基转移酶，以便将来用作生物催化剂。
