Ferredoxins as interchangeable redox components in support of MiaB, a radical S-adenosylmethionine methylthiotransferase

Arthur J. Arcinas, Stephanie J. Maiocco, Sean J. Elliott, Alexey Silakov, Squire J. Booker

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

MiaB is a member of the methylthiotransferase subclass of the radical S-adenosylmethionine (SAM) superfamily of enzymes, catalyzing the methylthiolation of C2 of adenosines bearing an N6-isopentenyl (i6A) group found at position 37 in several tRNAs to afford 2-methylthio-N6-(isopentenyl)adenosine (ms2i6A). MiaB uses a reduced [4Fe–4S]+ cluster to catalyze a reductive cleavage of SAM to generate a 5′-deoxyadenosyl 5′-radical (5′-dA•)—a required intermediate in its reaction—as well as an additional [4Fe–4S]2+ auxiliary cluster. In Escherichia coli and many other organisms, re-reduction of the [4Fe–4S]2+ cluster to the [4Fe–4S]+ state is accomplished by the flavodoxin reducing system. Most mechanistic studies of MiaBs have been carried out on the enzyme from Thermotoga maritima (Tm), which lacks the flavodoxin reducing system, and which is not activated by E. coli flavodoxin. However, the genome of this organism encodes five ferredoxins (TM0927, TM1175, TM1289, TM1533, and TM1815), each of which might donate the requisite electron to MiaB and perhaps to other radical SAM enzymes. The genes encoding each of these ferredoxins were cloned, and the associated proteins were isolated and shown to support turnover by Tm MiaB. In addition, TM1639, the ferredoxin-NADP+ oxidoreductase subunit α (NfnA) from Tm was overproduced and isolated and shown to provide electrons to the Tm ferredoxins during Tm MiaB turnover. The resulting reactions demonstrate improved coupling between formation of the 5′-dA• and ms2i6A production, indicating that only one hydrogen atom abstraction is required for the reaction.

Original languageEnglish (US)
Pages (from-to)267-282
Number of pages16
JournalProtein Science
Volume28
Issue number1
DOIs
StatePublished - Jan 1 2019

Fingerprint

Flavodoxin
Thermotoga maritima
Ferredoxins
S-Adenosylmethionine
Oxidation-Reduction
Adenosine
Escherichia coli
Bearings (structural)
Enzymes
Ferredoxin-NADP Reductase
Gene encoding
Electrons
Transfer RNA
Hydrogen
Genes
Atoms
Genome
Proteins

Keywords

  • ferredoxin
  • iron–sulfur cluster
  • methylthiolation
  • redox homeostasis
  • redox potential
  • S-adenosylmethionine

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Ferredoxins as interchangeable redox components in support of MiaB, a radical S-adenosylmethionine methylthiotransferase. / Arcinas, Arthur J.; Maiocco, Stephanie J.; Elliott, Sean J.; Silakov, Alexey; Booker, Squire J.

In: Protein Science, Vol. 28, No. 1, 01.01.2019, p. 267-282.

Research output: Contribution to journalArticle

Arcinas, Arthur J. ; Maiocco, Stephanie J. ; Elliott, Sean J. ; Silakov, Alexey ; Booker, Squire J. / Ferredoxins as interchangeable redox components in support of MiaB, a radical S-adenosylmethionine methylthiotransferase. In: Protein Science. 2019 ; Vol. 28, No. 1. pp. 267-282.
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abstract = "MiaB is a member of the methylthiotransferase subclass of the radical S-adenosylmethionine (SAM) superfamily of enzymes, catalyzing the methylthiolation of C2 of adenosines bearing an N6-isopentenyl (i6A) group found at position 37 in several tRNAs to afford 2-methylthio-N6-(isopentenyl)adenosine (ms2i6A). MiaB uses a reduced [4Fe–4S]+ cluster to catalyze a reductive cleavage of SAM to generate a 5′-deoxyadenosyl 5′-radical (5′-dA•)—a required intermediate in its reaction—as well as an additional [4Fe–4S]2+ auxiliary cluster. In Escherichia coli and many other organisms, re-reduction of the [4Fe–4S]2+ cluster to the [4Fe–4S]+ state is accomplished by the flavodoxin reducing system. Most mechanistic studies of MiaBs have been carried out on the enzyme from Thermotoga maritima (Tm), which lacks the flavodoxin reducing system, and which is not activated by E. coli flavodoxin. However, the genome of this organism encodes five ferredoxins (TM0927, TM1175, TM1289, TM1533, and TM1815), each of which might donate the requisite electron to MiaB and perhaps to other radical SAM enzymes. The genes encoding each of these ferredoxins were cloned, and the associated proteins were isolated and shown to support turnover by Tm MiaB. In addition, TM1639, the ferredoxin-NADP+ oxidoreductase subunit α (NfnA) from Tm was overproduced and isolated and shown to provide electrons to the Tm ferredoxins during Tm MiaB turnover. The resulting reactions demonstrate improved coupling between formation of the 5′-dA• and ms2i6A production, indicating that only one hydrogen atom abstraction is required for the reaction.",
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N2 - MiaB is a member of the methylthiotransferase subclass of the radical S-adenosylmethionine (SAM) superfamily of enzymes, catalyzing the methylthiolation of C2 of adenosines bearing an N6-isopentenyl (i6A) group found at position 37 in several tRNAs to afford 2-methylthio-N6-(isopentenyl)adenosine (ms2i6A). MiaB uses a reduced [4Fe–4S]+ cluster to catalyze a reductive cleavage of SAM to generate a 5′-deoxyadenosyl 5′-radical (5′-dA•)—a required intermediate in its reaction—as well as an additional [4Fe–4S]2+ auxiliary cluster. In Escherichia coli and many other organisms, re-reduction of the [4Fe–4S]2+ cluster to the [4Fe–4S]+ state is accomplished by the flavodoxin reducing system. Most mechanistic studies of MiaBs have been carried out on the enzyme from Thermotoga maritima (Tm), which lacks the flavodoxin reducing system, and which is not activated by E. coli flavodoxin. However, the genome of this organism encodes five ferredoxins (TM0927, TM1175, TM1289, TM1533, and TM1815), each of which might donate the requisite electron to MiaB and perhaps to other radical SAM enzymes. The genes encoding each of these ferredoxins were cloned, and the associated proteins were isolated and shown to support turnover by Tm MiaB. In addition, TM1639, the ferredoxin-NADP+ oxidoreductase subunit α (NfnA) from Tm was overproduced and isolated and shown to provide electrons to the Tm ferredoxins during Tm MiaB turnover. The resulting reactions demonstrate improved coupling between formation of the 5′-dA• and ms2i6A production, indicating that only one hydrogen atom abstraction is required for the reaction.

AB - MiaB is a member of the methylthiotransferase subclass of the radical S-adenosylmethionine (SAM) superfamily of enzymes, catalyzing the methylthiolation of C2 of adenosines bearing an N6-isopentenyl (i6A) group found at position 37 in several tRNAs to afford 2-methylthio-N6-(isopentenyl)adenosine (ms2i6A). MiaB uses a reduced [4Fe–4S]+ cluster to catalyze a reductive cleavage of SAM to generate a 5′-deoxyadenosyl 5′-radical (5′-dA•)—a required intermediate in its reaction—as well as an additional [4Fe–4S]2+ auxiliary cluster. In Escherichia coli and many other organisms, re-reduction of the [4Fe–4S]2+ cluster to the [4Fe–4S]+ state is accomplished by the flavodoxin reducing system. Most mechanistic studies of MiaBs have been carried out on the enzyme from Thermotoga maritima (Tm), which lacks the flavodoxin reducing system, and which is not activated by E. coli flavodoxin. However, the genome of this organism encodes five ferredoxins (TM0927, TM1175, TM1289, TM1533, and TM1815), each of which might donate the requisite electron to MiaB and perhaps to other radical SAM enzymes. The genes encoding each of these ferredoxins were cloned, and the associated proteins were isolated and shown to support turnover by Tm MiaB. In addition, TM1639, the ferredoxin-NADP+ oxidoreductase subunit α (NfnA) from Tm was overproduced and isolated and shown to provide electrons to the Tm ferredoxins during Tm MiaB turnover. The resulting reactions demonstrate improved coupling between formation of the 5′-dA• and ms2i6A production, indicating that only one hydrogen atom abstraction is required for the reaction.

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