39] Diphthamide: In Vitro biosynthesis

Abstract

This chapter focuses on in vitro biosynthesis of diphthamide. Elongation factor 2 (EF-2), the protein synthesis translocase enzyme, possesses a unique amino acid called “diphthamide,” a derivative of histidine with the proposed structure 2-[3-carboxyamido-3 (trimethylammonio)propyl]histidine. Diphthamide is synthesized by a complex posttranslational modification of a histidine residue of EF-2. Hybrids between wild-type and mutant cells produce only sensitive EF-2. These mutants possess mutations that affect the posttranslational biosynthesis of diphthamide, referred to as modification, or MOD – , mutants. Complementation analysis has revealed that there are at least 3 distinct complementation groups of MOD – mutants that can be isolated from CHO-K1 cells. The diphthamide residue in the EF-2 of cells of these 3 complementation groups lacks different portions of the 4-carbon side chain. When exposed to normal modification enzymes in a cell-cell hybrid, the modification of the diphthamide is completed. The diphthamide in EF-2 from the complementation group 1 (CG-1), apparently lacks the three methyl residues of the trimethylamino group on the side chain.