Yeast gene KTI13 (alias DPH8) operates in the initiation step of diphthamide synthesis on elongation factor 2

Abstract

In yeast, Elongator-dependent tRNA modifications are regulated by the Kti11•Kti13 dimer and hijacked for cell killing by zymocin, a tRNase ribotoxin. Kti11 (alias Dph3) also controls modification of elongation factor 2 (EF2) with diphthamide, the target for lethal ADP-ribosylation by diphtheria toxin (DT). Diphthamide formation on EF2 involves four biosynthetic steps encoded by the DPH1-DPH7 network and an ill-defined KTI13 function. On further examining the latter gene in yeast, we found that kti13Δ null-mutants maintain unmodified EF2 able to escape ADP-ribosylation by DT and to survive EF2 inhibition by sordarin, a diphthamide-dependent antifungal. Consistently, mass spectrometry shows kti13Δ cells are blocked in proper formation of amino-carboxyl-propyl-EF2, the first diphthamide pathway intermediate. Thus, apart from their common function in tRNA modification, both Kti11/Dph3 and Kti13 share roles in the initiation step of EF2 modification. We suggest an alias KTI13/DPH8 nomenclature indicating dual-functionality analogous to KTI11/DPH3.

Functional Integrity of Radical SAM Enzyme Dph1•Dph2 Requires Non-Canonical Cofactor Motifs with Tandem Cysteines

“…The latter antifungal stalls ribosomes and blocks protein synthesis in a fashion unrelated to DT but also dependent on the diphthamide décor on eEF2 [38][39][40]. (A) Simplified diphthamide pathway with DPH1 and DPH2 genes required to initiate ACP formation [21]; subsequent DPH products and steps for the completion of diphthamide synthesis [14][15][16][17] are not detailed. Diphthamide can be hijacked to induce cell death (skull-crossbones) either by DT for lethal ADP-ribosylation of eEF2 or sordarin (sor), which in a complex with the décor on eEF2 stalls ribosomes [40].…”

“…Dph1•Dph2 processes SAM into a non-canonical 3-amino-3-carboxy-propyl Biomolecules 2024, 14, 470 2 of 14 (ACP) radical used for the modification of eukaryotic translation elongation factor 2 (eEF2) with diphthamide [11][12][13]. Diphthamide is formed via a dedicated synthesis network (i.e., Dph1-Dph8) on a conserved histidine of eEF2 in eukaryotes (His-699 in budding yeast Saccharomyces cerevisiae and His-715 in Homo sapiens) and EF2 in some archaea [14][15][16][17]. Importantly, human patients deficient in diphthamide-modified eEF2 display symptoms of a neurodevelopmental disorder known as diphthamide deficiency syndrome (DDS) [18][19][20][21][22] and diphtheria toxin (DT) from Corynebacterium diphtheriae attacks the diphthamide décor on eEF2 by ADP-ribosylation to block mRNA translation in infected host cells [23].…”

“…Importantly, human patients deficient in diphthamide-modified eEF2 display symptoms of a neurodevelopmental disorder known as diphthamide deficiency syndrome (DDS) [18][19][20][21][22] and diphtheria toxin (DT) from Corynebacterium diphtheriae attacks the diphthamide décor on eEF2 by ADP-ribosylation to block mRNA translation in infected host cells [23]. Yeast diphthamide mutants (i.e., dph1∆-dph8∆) are resistant to growth inhibition by DT, which is why the toxin is a valuable molecular tool to tell diphthamide proficiency from deficiency in vivo [17,24].…”

Functional Integrity of Radical SAM Enzyme Dph1•Dph2 Requires Non-canonical Cofactor Motifs with Tandem Cysteines

“…2 translation elongation factor 2 (eEF2) with diphthamide [11][12][13]. Diphthamide is formed by a dedicated synthesis network (i.e., Dph1-Dph8) on a conserved histidine of eEF2 in eukaryotes (His-699 in budding yeast Saccharomyces cerevisiae and His-715 in Homo sapiens) and EF2 in some archaea [14][15][16][17]. Importantly, human patients deficient in diphthamide-modified eEF2 display symptoms of a neurodevelopmental disorder known as diphthamide deficiency syndrome (DDS) [18][19][20][21][22] and diphtheria toxin (DT) from Corynebacterium diphtheriae attacks the diphthamide décor on eEF2 by ADP-ribosylation to block mRNA translation in infected host cells [23].…”

“…Importantly, human patients deficient in diphthamide-modified eEF2 display symptoms of a neurodevelopmental disorder known as diphthamide deficiency syndrome (DDS) [18][19][20][21][22] and diphtheria toxin (DT) from Corynebacterium diphtheriae attacks the diphthamide décor on eEF2 by ADP-ribosylation to block mRNA translation in infected host cells [23]. Yeast diphthamide mutants (i.e., dph1Δ-dph8Δ) are resistant to eEF2 and growth inhibition by DT, which is why the toxin is a valuable molecular tool to tell diphthamide proficiency from deficiency in vivo [17,24].…”