TY - JOUR
T1 - Methionine Adenosyltransferase Engineering to Enable Bioorthogonal Platforms for AdoMet-Utilizing Enzymes
AU - Huber, Tyler D.
AU - Clinger, Jonathan A.
AU - Liu, Yang
AU - Xu, Weijun
AU - Miller, Mitchell D.
AU - Phillips, George N.
AU - Thorson, Jon S.
N1 - Publisher Copyright:
© 2020 American Chemical Society.
PY - 2020/3/20
Y1 - 2020/3/20
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85082145099&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85082145099&partnerID=8YFLogxK
U2 - 10.1021/acschembio.9b00943
DO - 10.1021/acschembio.9b00943
M3 - Article
C2 - 32091873
AN - SCOPUS:85082145099
SN - 1554-8929
VL - 15
SP - 695
EP - 705
JO - ACS Chemical Biology
JF - ACS Chemical Biology
IS - 3
ER -