Inactivation of E. coli pyruvate formate-lyase: Role of AdhE and small molecules

Abstract

E. coli AdhE has been reported to harbor three distinct enzymatic activities: alcohol dehydrogenase, acetaldehyde-CoA dehydrogenase, and pyruvate formate-lyase (PFL) deactivase. Herein we report on the cloning, expression, and purification of E. coli AdhE, and the re-investigation of its purported enzymatic activities. While both the alcohol dehydrogenase and acetaldehyde-CoA dehydrogenase activities were readily detectible, we were unable to obtain any evidence for catalytic deactivation of PFL by AdhE, regardless of whether the reported cofactors for deactivation (Fe(II), NAD, and CoA) were present. Our results demonstrate that AdhE is not a PFL deactivating enzyme. We have also examined the potential for deactivation of active PFL by small-molecule thiols. Both β-mercaptoethanol and dithiothreitol deactivate PFL efficiently, with the former providing quite rapid deactivation. PFL deactivated by these thiols can be reactivated, suggesting that this deactivation is non-destructive transfer of an H atom equivalent to quench the glycyl radical.

Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity

“…For HPAD, it has been proposed that the [4Fe-4S] clusters are redox active and could act to reductively quench the Gly radical by electron transfer, returning the enzyme to the inactive state (18). Efficient, reversible reduction of the Gly radical has been observed in vitro in PFL in the presence of DTT or β-mercaptoethanol (29), suggesting that Gly radical reduction could be a means of preventing permanent damage to the GRE by quenching the radical before O 2 -catalyzed backbone cleavage can occur. However, no data are available to support the existence of this type of protective mechanism in vivo.…”

Pyruvate Formate-lyase, Evidence for an Open Conformation Favored in the Presence of Its Activating Enzyme

“…1); however, it is followed immediately by the time-dependent loss of the glycyl radical. The loss of the glycyl radical is likely a result of nondestructive quenching by the high concentrations of DTT in the assays, as we have reported previously (29); it is, however, dramatically faster in the presence of stoichiometric PFL-AE (t1 ⁄ 2 ϳ 90 min) than in the presence of catalytic amounts of PFL-AE (t1 ⁄ 2 Ն 24 h). The observation that PFL is more prone to reductive quenching when activated with a stoichiometric, rather than catalytic, quantity of PFL-AE suggests that the interaction with PFL-AE favors an alternate conformation of PFL in which Gly-734 is more solvent-exposed; we heretofore refer to this as the open conformation of PFL.…”

“…As expected, under these conditions the PFL was fully activated much more rapidly than when catalytic amounts of PFL-AE were used; however, the rapid and complete activation was followed by a steady loss in the amount of glycyl radical present. PFL that had lost its glycyl radical during the course of an extended incubation with stoichiometric PFL-AE could be reactivated by subjecting it again to activation conditions; this ability to regenerate the glycyl radical lost upon long incubation demonstrates that the glycyl radical is lost by nondestructive quenching, most likely by the high concentrations of DTT in the buffer, which we have previously shown to quench the PFL glycyl radical nondestructively (29). These results together provide evidence that at higher concentrations of PFL-AE, the Gly-734 radical of PFL has a greater probability of undergoing nondestructive quenching; this observation suggests that the presence of PFL-AE favors an open conformation of PFL in which Gly-734 is more solvent-exposed.…”

“…AdoMet was synthesized according to the procedure described previously (24). PFL was overexpressed in Escherichia coli strain BL21(DE3)plysS transformed with pKK/pfl and purified as described previously (17,29). PFL-AE was overexpressed in E. coli strain BL21(DE3)plysS transformed with pCAL-n-AE and purified as described previously (23,30).…”

“…Activated PFL (1 l) was added into buffer (99 l) containing 100 mM Tris-HCl, pH 7.6, and 100 mM KCl. An aliquot of this diluted sample (5 l) was taken out and mixed with 795 l of the coupling assay mix, and the rate of PFL turnover was determined spectrophotometrically as described previously (1,29).…”