Methods for Studying the Radical SAM Enzymes in Diphthamide Biosynthesis

Methods Enzymol. 2018:606:421-438. doi: 10.1016/bs.mie.2018.04.001.

Abstract

Diphthamide is a unique posttranslational modification on translation elongation factor 2 (EF2) in archaea and eukaryotes. Biosynthesis of diphthamide was proposed to involve four steps. The first step is a CC bond forming reaction catalyzed by unique radical S-adenosylmethionine (SAM) enzymes. Classical radical SAM enzymes use SAM and [4Fe-4S] clusters to generate a 5'-deoxyadenynal radical and catalyze numerous reactions. Radical SAM enzymes in diphthamide biosynthesis cleave a different CS bond in SAM to generate a 3-amino-3-carboxypropyl radical and modify a histidine residue of substrate protein EF2. Here, we describe our investigations on these unique radical SAM enzymes, including the preparation, characterization, and activity assays we have developed.

Keywords: Diphthamide; Fe–S cluster; Radical SAM enzymes.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkyl and Aryl Transferases / isolation & purification
  • Alkyl and Aryl Transferases / metabolism*
  • Archaeal Proteins / isolation & purification
  • Archaeal Proteins / metabolism*
  • Enzyme Assays / methods*
  • Histidine / analogs & derivatives*
  • Histidine / biosynthesis
  • Peptide Elongation Factor 2 / metabolism
  • Protein Processing, Post-Translational
  • Pyrococcus horikoshii
  • S-Adenosylmethionine / metabolism*

Substances

  • Archaeal Proteins
  • Peptide Elongation Factor 2
  • Histidine
  • diphthamide
  • S-Adenosylmethionine
  • Alkyl and Aryl Transferases