title: 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 cleave homolytically 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 a 3-amino-3-carboxylpropyl radical. We here show 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 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 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 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 provides an illustration of the versatile reactivity of Fe-S clusters.
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University , Ithaca, New York 14853, United States.
authors.forename: Min
authors.initials: M
authors.lastname: Dong
authors.affiliation: Department of Chemistry, Northwestern University , Evanston, Illinois 60208, United States.|Department of Applied Biochemistry and Food Science, Saga University , Saga 840-8502, Japan.
authors.forename: Masaki
authors.identifier: 0000-0002-8881-0600
authors.initials: M
authors.lastname: Horitani
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University , Ithaca, New York 14853, United States.
authors.forename: Boris
authors.initials: B
authors.lastname: Dzikovski
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University , Ithaca, New York 14853, United States.
authors.forename: Jack H
authors.identifier: 0000-0003-4288-2585
authors.initials: JH
authors.lastname: Freed
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University , Ithaca, New York 14853, United States.
authors.forename: Steven E
authors.initials: SE
authors.lastname: Ealick
authors.affiliation: Department of Chemistry, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Brian M
authors.identifier: 0000-0002-3100-0746
authors.initials: BM
authors.lastname: Hoffman
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Cornell University , Ithaca, New York 14853, United States.
authors.forename: Hening
authors.identifier: 0000-0002-0255-2701
authors.initials: H
authors.lastname: Lin
mesh_terms: D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D043902:Pyrococcus horikoshii / Q000201:enzymology; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D005609:Free Radicals; D007506:Iron-Sulfur Proteins; D006639:Histidine; C027527:diphthamide; D012436:S-Adenosylmethionine