Chemical and biological reduction of the radical SAM enzyme CPH<inf>4</inf> synthase

Nathan A. Bruender, Anthony P. Young, Vahe Bandarian

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

The radical S-adenosyl-l-methionine (SAM) superfamily is a large and growing group of enzymes that conduct complex radical-mediated transformations. A one-electron reduction of SAM via the +1 state of the cubane [4Fe-4S] cluster generates a 5′-deoxyadenosyl radical, which initiates turnover. The [4Fe-4S] cluster must be reduced from its resting +2 state to the catalytically active +1 oxidation state by an electron. In practice, dithionite or the Escherichia coli flavodoxin (EcFldA)/ferredoxin (flavodoxin):NADP<sup>+</sup> oxidoreductase (Fpr)/NADPH system is used. Herein, we present a systematic investigation of the reductive activation of the radical SAM enzyme CDG synthase (BsQueE) from Bacillus subtilis comparing biological and chemical reductants. These data show that either of the flavodoxin homologues encoded by the B. subtilis genome, BsYkuN or BsYkuP, as well as a series of small molecule redox mediators, supports BsQueE activity. With dithionite as a reductant, the activity of BsQueE is ∼75-fold greater in the presence of BsYkuN and BsYkuP compared to that in the presence of dithionite alone. By contrast, EcFldA supports turnover to ∼10-fold greater levels than dithionite alone under the same conditions. Comparing the ratio of the rate of turnover to the apparent binding constant for the flavodoxin homologues reveals 10- and 240-fold preferences for BsYkuN over BsYkuP and EcFldA, respectively. The differential activation of the enzyme cannot be explained by the abortive cleavage of SAM. We conclude from these observations that the differential activation of BsQueE by Fld homologues may reside in the details of the interaction between the flavodoxin and the radical SAM enzyme.

Original languageEnglish (US)
Pages (from-to)2903-2910
Number of pages8
JournalBiochemistry
Volume54
Issue number18
DOIs
StatePublished - May 12 2015

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Flavodoxin
Dithionite
Methionine
Enzymes
Chemical activation
Reducing Agents
Bacillus subtilis
Ferredoxin-NADP Reductase
Electrons
Ferredoxins
Enzyme Activation
Bacilli
NADP
Escherichia coli
Oxidation-Reduction
Genes
Genome
Oxidation
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

Chemical and biological reduction of the radical SAM enzyme CPH<inf>4</inf> synthase. / Bruender, Nathan A.; Young, Anthony P.; Bandarian, Vahe.

In: Biochemistry, Vol. 54, No. 18, 12.05.2015, p. 2903-2910.

Research output: Contribution to journalArticle

Bruender, Nathan A. ; Young, Anthony P. ; Bandarian, Vahe. / Chemical and biological reduction of the radical SAM enzyme CPH<inf>4</inf> synthase. In: Biochemistry. 2015 ; Vol. 54, No. 18. pp. 2903-2910.
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