The asymmetric function of Dph1-Dph2 heterodimer in diphthamide biosynthesis.
J Biol Inorg Chem
; 24(6): 777-782, 2019 09.
Article
in En
| MEDLINE
| ID: mdl-31463593
ABSTRACT
Diphthamide, the target of diphtheria toxin, is a post-translationally modified histidine residue found in archaeal and eukaryotic translation elongation factor 2 (EF2). In the first step of diphthamide biosynthesis, a [4Fe-4S] cluster-containing radical SAM enzyme, Dph1-Dph2 heterodimer in eukaryotes or Dph2 homodimer in archaea, cleaves S-adenosylmethionine and transfers the 3-amino-3-carboxypropyl group to EF2. It was demonstrated previously that for the archaeal Dph2 homodimer, only one [4Fe-4S] cluster is necessary for the in vitro activity. Here, we demonstrate that for the eukaryotic Dph1-Dph2 heterodimer, the [4Fe-4S] cluster-binding cysteine residues in each subunit are required for diphthamide biosynthesis to occur in vivo. Furthermore, our in vitro reconstitution experiments with Dph1-Dph2 mutants suggested that the Dph1 cluster serves a catalytic role, while the Dph2 cluster facilitates the reduction of the Dph1 cluster by the physiological reducing system Dph3/Cbr1/NADH. Our results reveal the asymmetric functional roles of the Dph1-Dph2 heterodimer and may help to understand how the Fe-S clusters in radical SAM enzymes are reduced in biology.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Archaeal Proteins
/
Histidine
/
Iron-Sulfur Proteins
Language:
En
Journal:
J Biol Inorg Chem
Journal subject:
BIOQUIMICA
Year:
2019
Document type:
Article
Affiliation country:
United States