Reconstitution of diphthine synthase activity in vitro.
Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 (EF2). Although diphthamide modification was discovered three decades ago, in vitro reconstitution of diphthamide biosynthesis using purified proteins has not been reported. The proposed biosynthesis pathway of diphthamide involves three steps. Our laboratory has recently showed that in Pyrococcus horikoshii (P. horikoshii), the first step uses a [4Fe-4S] enzyme PhDph2 to generate a 3-amino-3-carboxypropyl radical from S-adenosyl-L-methionine (SAM) to form a C−C bond. The second step is the trimethylation of an amino group to form the diphthine intermediate. This step is catalyzed by a methyltransferase called diphthine synthase or Dph5. Here we report the in vitro reconstitution of the second step using P. horikoshii Dph5 (PhDph5). Our results demonstrate that PhDph5 is sufficient to catalyze the mono-, di-, and trimethylation of P. horikoshii EF2 (PhEF2). Interestingly, the trimethylated product from the PhDph5-catalyzed reaction can easily eliminate the trimethylamino group. The potential implication of this unexpected finding on the diphthamide biosynthesis pathway is discussed.
Zhu
Xuling
X
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
Kim
Jungwoo
J
Su
Xiaoyang
X
Lin
Hening
H
D019843:Archaeal Proteins / Q000378:metabolism*; D053898:Biosynthetic Pathways; D006639:Histidine / Q000031:analogs & derivatives* / Q000378:metabolism; D008780:Methyltransferases / Q000378:metabolism*; D020652:Peptide Elongation Factor 2 / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D043902:Pyrococcus horikoshii / Q000201:enzymology* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D019843:Archaeal Proteins; D020652:Peptide Elongation Factor 2; D006639:Histidine; C027527:diphthamide; C027529:diphthine; D008780:Methyltransferases