Chemical and Biological Reduction of the Radical SAM Enzyme 7-Carboxy-7-deazaguanine [corrected] Synthase - PubMed (original) (raw)

Chemical and Biological Reduction of the Radical SAM Enzyme 7-Carboxy-7-deazaguanine [corrected] Synthase

Nathan A Bruender et al. Biochemistry. 2015.

Erratum in

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(+) 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.

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Figures

Figure 1

Figure 1

Time course of _Bs_QueE reaction under various reducing conditions. Activity was monitored by measuring CDG formation._Bs_QueE activity was measured in the presence 10 mM dithionite (upside-down triangle) or 2 μM Fpr/2 mM NADPH with 5 μM_Bs_YkuN (circle), 5 μM Fpr/2 mM NADPH with 20 μM _Bs_YkuP (triangle), or 5 μM Fpr/2 mM NADPH with 20 μM _Ec_FldA (diamond). Assays where Fpr or NADPH (square) were omitted served as negative controls.

Figure 2

Figure 2

_Bs_QueE activity is enhanced by increasing the flavodoxin homologs [_Ec_FldA(diamonds)], [_Bs_YkuN (circles)], or [_Bs_YkuP (triangles)] concentration when the electrons are supplied by either NADPH via Fpr (A) or dithionite (B). To confirm Fld reduction by NADPH/Fpr was saturating, the rate of CDG formation dependent on [Fld] was measured at 1 μM (closed) and 5 μM (open) Fpr for _Bs_YkuN and 5 μM (closed) and 20 μM (open) for _Bs_YkuP and _Ec_FldA (A). Depicted are representative data sets for each experiment. The data were fit using Eqn 1 and the _V_max (_k_CDG) ant_K_Fld are reported in Table 1.

Figure 3

Figure 3

_Bs_QueE activity is enhanced by increasing methyl viologen concentration when electrons are supplied by dithionite. The data were fit using Eqn. 2 to determine_k_CDG. The activity of _Bs_QueE was 0.2 min−1 in the absence of methyl viologen.

Figure 4

Figure 4

Velocity of CDG formation (circles) or dAdo formation (squares) as a function of reduction potential of redox mediator used in the activity assay. Dithionite was the reductant used in all of the reactions. The redox mediators used were methyl viologen (Em= −0.446 V), benzyl viologen (Em= −0.359 V), neutral red (Em= −0.325 V), and lissamine blue BF (Em= −0.276 V, pH 7.5). The dashed lines represent the rates of formation for CDG (–––) and dAdo (---), 0.2 min−1 and 0.08 min−1respectively, when _Bs_QueE was activated by dithionite alone.

Scheme 1

Scheme 1

Reductive cleavage of SAM.

Scheme 2

Scheme 2

Electron flow for [4Fe-4S] cluster reduction.a a The scheme shows the flow of electrons and does not imply absolute stoichiometry.

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