Substrate-Dependent Cleavage Site Selection by Unconventional Radical S-Adenosylmethionine Enzymes in Diphthamide Biosynthesis

Abstract

S -adenosylmethionine (SAM) has a sulfonium ion with three distinct C-S bonds. Conventional radical SAM enzymes use a [4Fe–4S] cluster to homolytically cleave the C5′,adenosine-S bond of SAM to generate a 5′-deoxyadenosyl radical, which catalyzes various downstream chemical reactions. Radical SAM enzymes involved in diphthamide biosynthesis, such as Pyrococcus horikoshii Dph2 (PhDph2) and yeast Dph1-Dph2 instead cleave the Cγ,Met–S bond of methionine to generate the 3-amino-3-carboxylpropyl radical. We here show that radical SAM enzymes can be tuned to cleave the third C-S bond to the sulfonium sulfur by changing the structure of SAM. With a decarboxyl SAM analogue (dc-SAM), PhDph2 cleaves the Cmethyl–S bond, forming 5′-deoxy-5′-(3-aminopropylthio) adenosine (dAPTA, 1). The methyl cleavage activity, like the cleavage of the other two C-S bonds, is dependent on the presence of a [4Fe-4S]+ cluster. Electron-nuclear double resonance (ENDOR) and mass spectroscopy data suggests that mechanistically, one of the S atoms in the [4Fe-4S] cluster captures the methyl group from dc-SAM, forming a distinct EPR-active intermediate, which can transfer the methyl group to nucleophiles such as dithiothreitol. This reveals that the [4Fe-4S] cluster in a radical SAM enzyme can be tuned to cleave any one of the three bonds to the sulfonium sulfur of SAM or analogues, and is the first demonstration that a radical SAM enzyme could switch from an Fe-based one electron transfer reaction to a S-based two electron transfer reaction in a substrate-dependent manner. This study thus provides a remarkable illustration of the versatile reactivity of Fe-S clusters.

C–C bond forming radical SAM enzymes involved in the construction of carbon skeletons of cofactors and natural products

“…In a separate report, PhDph2 was also shown to catalyze methyl transfer reaction from decarboxy-or deamino-SAM analogs onto one of the S atoms of the [4Fe–4S] cluster. 155 Together, these observations revealed the electron rich and potentially nucleophilic nature of the PhDph2 [4Fe–4S] cluster. The unique reactivity of the PhDph2 [4Fe–4S] cluster is particularly interesting considering the distinct selectivity of SAM cleavage in Dph2 vs. 5′-dA • forming radical SAM enzymes, and should be investigated in more detail in the future.…”

Revisiting the methionine salvage pathway and its paralogues

“…Homologues of Tsr3 (DUF367 family proteins) are widespread in archaea and present in some bacteria as well (Burroughs and Aravind, 2014). In the same way, synthesis of the modified histidine residue diphthamide in archaeal and eukaryotic elongation factor 2 (eEF-2) uses transfer of this group in a non-standard radical reaction (Dong et al, 2017a). It may also be worth investigating whether there exist protein modifications (or modifications of metabolites other than polyamines) that use aminopropyl rather than methyl groups.…”

“…This is reminiscent of the group transfers we have previously discussed (see the biochemical rationale for the standard MSP). To be sure, we have noticed that diphthamide biosynthesis generated MTA via a radical reaction that was at odds with those mediated by the other well-known radical AdoMet enzymes (Dong et al, 2017a). Further, in a few reactions, AdoMet is recycled intact, as is a coenzyme.…”

Importance of diphthamide modified EF2 for translational accuracy and competitive cell growth in yeast

“…Indeed, its formation is conserved from archaea to yeast and man and in eukaryotes, involves a multi-step biosynthetic pathway ( Fig 1 ) that is encoded by the DPH1-DPH7 gene network [ 12 – 19 ]. Starting with the addition of a 3-amino-3-carboxypropyl (ACP) radical from S-adenosyl-methionine (SAM) to the imidazole ring of the crucial histidine in EF2 (yeast: His-699; humans: His-715), step one of the pathway ( Fig 1 ) engages Fe/S enzyme (Dph1•Dph2) chemistry, electron transfer (Dph3) and a putative J-type chaperone (Dph4) to form ACP-modified EF2 [ 20 – 27 ]. Multiple methylation of this first intermediate by a methylase (Dph5) generates methyl-diphthine [ 28 – 31 ] ( Fig 1 ).…”