Open AccessFluorescent Probes for Monitoring Serine Ubiquitination
Author(s) -
Kedar Puvar,
Aya M. Saleh,
Ryan Curtis,
Yiyang Zhou,
Prasanth R. Nyalapatla,
Jiaqi Fu,
Alexander R. Rovira,
Yitzhak Tor,
ZhaoQing Luo,
Arun K. Ghosh,
Mary J. Wirth,
Jean Chmielewski,
Tamara L. KinzerUrsem,
Chittaranjan Das
Publication year - 2020
Publication title -
biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.43
H-Index - 253
eISSN - 1520-4995
pISSN - 0006-2960
DOI - 10.1021/acs.biochem.0c00067
Subject(s) - effector , ubiquitin , serine , nad+ kinase , enzyme , biochemistry , chemistry , legionella pneumophila , residue (chemistry) , biology , microbiology and biotechnology , bacteria , genetics , gene
In a radical departure from the classical E1-E2-E3 three-enzyme mediated ubiquitination of eukaryotes, the recently described bacterial enzymes of the SidE family of Legionella pneumophila effectors utilize NAD + to ligate ubiquitin onto target substrate proteins. This outcome is achieved via a two-step mechanism involving (1) ADP ribosylation of ubiquitin followed by (2) phosphotransfer to a target serine residue. Here, using fluorescent NAD + analogues as well as synthetic substrate mimics, we have developed continuous assays enabling real-time monitoring of both steps of this mechanism. These assays are amenable to biochemical studies and high-throughput screening of inhibitors of these effectors, and the discovery and characterization of putative enzymes similar to members of the SidE family in other organisms. We also show their utility in studying enzymes that can reverse and inhibit this post-translational modification.