Steve Ealick's Research Group
Pyrococcus horikoshii Dph2
3LZD PhDph2 with the iron sulfur cluster
Archaeal and eukaryotic translation elongation factor 2 (EF-2) contain a unique posttranslationally modified histidine residue called “diphthamide”, the target of diphtheria toxin.The first step of diphthamide biosynthesis in archaea uses a novel enzyme, Dph2, that contains an iron-sulfur cluster. Biochemical data suggest that unlike known radical SAM enzymes, Pyrococcus horikoshi Dph2 (PhDph2) does not form 5´-deoxyadenosyl radicals. Instead, it breaks the other C-S bond of SAM and transfers the 3-amino-3-carboxylpropyl group to EF-2, possibly via a radical mechanism. This work suggests that archaeal Dph2 represents a novel SAM-dependent [4Fe-4S]-containing enzyme that catalyzes unprecedented chemistry.
Each PhDph2 monomer consists of three domains with all three domains sharing the same overall fold, suggesting that the monomer is the result of a gene triplication. Three absolutely conserved cysteine residues (Cys59, Cys163 and Cys287), which are far away from each other in the primary sequence, are clustered together in the center of each PhDph2 monomer and each of these cysteine residues comes from a different domain.
|PhDph2 is a homodimer in which domain 1 of one monomer and domain 3 of the adjacent monomer form the dimer interface, creating an extended nine-stranded β-
dpH2 appears to be part of the radical SAM (S-adenosylmethionine) superfamily. The cysteines from which the [4Fe-4S] cluster is formed are located in separate structural domains, which may possibly result from gene triplication, and are separated by more than 100 amino acid residues in the primary sequence.
Zhang Y, Zhu X, Torelli AT, Lee M, Dzikovski B, Koralewski RM, Wang E, Freed J, Krebs C, Ealick SE, Lin H. Diphthamide biosynthesis requires an organic radical generated by an iron–sulphur enzyme. Nature 465:891-896 (2010). Abstract. PubMed; associated Nature News and View article; associated article in Chemical & Engineering News; associated note in Nature Chemical Biology(scroll down to "Metalloenzymes: A road less traveled").. Nature Chemical Biology recently described this radical new SAM mechanism as one of the key developments in chemical biology in the past decade.
Zhu X, Dzikovski B, Su X, Torelli AT, Zhang Y, Ealick SE, Freed JH, and Lin H. Mechanistic Understanding of Pyrococcus horikoshii Dph2, a [4Fe-4S] Enzyme Required for Diphthamide Biosynthesis. Mol. Biosyst. (in press) 2010). Abstract. PubMed