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.
|Number of pages||11|
|Journal||ACS Chemical Biology|
|State||Published - Mar 20 2020|
Bibliographical noteFunding Information:
This work was supported in part by NIH R01 GM115261 (J.S.T. and G.N.P.), the NIH Protein Structure Initiative (U01 GM098248, G.N.P.), NIH GM109456 (G.N.P.), NIH T32 GM008280 (J.A.C.), the University of Kentucky College of Pharmacy, the National Center for Advancing Translational Sciences (UL1TR000117 and UL1TR001998), and the National Science Foundation BioXFEL STC (1231306; G.N.P.). We also thank the University of Kentucky Mass Spectrometry Facility (ASTeCC) for HRMS support and the staff at the LS-CAT and GM/CA beamline at the Advanced Photo Source for help in conducting trial attempts and collecting the diffraction data. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006).
© 2020 American Chemical Society.
ASJC Scopus subject areas
- Molecular Medicine