Methionine Adenosyltransferase Engineering to Enable Bioorthogonal Platforms for AdoMet-Utilizing Enzymes
Tyler D. Huber, Jonathan A. Clinger, Yang Liu, Weijun Xu, Mitchell D. Miller, George N. Phillips, Jon S. Thorson
Abstract
The structural conservation among methyltransferases (MTs) and MT functional redundancy is a major challenge to the cellular study of individual MTs. As a first step toward the development of an alternative biorthogonal platform for MTs and other AdoMet-utilizing enzymes, we describe the evaluation of 38 human methionine adenosyltransferase II-α (hMAT2A) mutants in combination with 14 non-native methionine analogues to identify suitable bioorthogonal mutant/analogue pairings. Enabled by the development and implementation of a hMAT2A high-throughput (HT) assay, this study revealed hMAT2A K289L to afford a 160-fold inversion of the hMAT2A selectivity index for a non-native methionine analogue over the native substrate l-Met. Structure elucidation of K289L revealed the mutant to be folded normally with minor observed repacking within the modified substrate pocket. This study highlights the first example of exchanging l-Met terminal carboxylate/amine recognition elements within the hMAT2A active-site to enable non-native bioorthgonal substrate utilization. Additionally, several hMAT2A mutants and l-Met substrate analogues produced AdoMet analogue products with increased stability. As many AdoMet-producing (e.g., hMAT2A) and AdoMet-utlizing (e.g., MTs) enzymes adopt similar active-site strategies for substrate recognition, the proof of concept first generation hMAT2A engineering highlighted herein is expected to translate to a range of AdoMet-utilizing target enzymes.