Pyruvate formate lyase activating enzyme (PFL-AE) is a member of the Radical SAM superfamily that functions by catalyzing the formation of the Gly734 radical of PFL.
Characterization using rapid freeze-quenching to trap a catalytically competent intermediate and characterization of the intermediate by electron paramagnetic resonance, (13)C, and (57)Fe electron nuclear double-resonance spectroscopies reveals that the intermediate contains an organometallic center in which the 5' carbon of a SAM-derived deoxyadenosyl moiety forms a bond with the unique iron site of the [4Fe-4S] cluster.
Vey JL, Yang J, Li M, Broderick WE, Broderick JB, Drennan CL
Structural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme
▸ Abstract
Pyruvate formate-lyase activating enzyme generates a stable and catalytically essential glycyl radical on G(734) of pyruvate formate-lyase via the direct, stereospecific abstraction of a hydrogen atom from pyruvate formate-lyase. The activase performs this remarkable feat by using an iron-sulfur cluster and S-adenosylmethionine (AdoMet), thus placing it among the AdoMet radical superfamily of enzymes. We report here structures of the substrate-free and substrate-bound forms of pyruvate formate-lyase-activating enzyme, the first structures of an AdoMet radical activase. To obtain the substrate-bound structure, we have used a peptide substrate, the 7-mer RVSGYAV, which contains the sequence surrounding G(734). Our structures provide fundamental insights into the interactions between the activase and the G(734) loop of pyruvate formate-lyase and provide a structural basis for direct and stereospecific H atom abstraction from the buried G(734) of pyruvate formate-lyase.
Proc Natl Acad Sci U S A
2008;105(42):16137-16141
| PubMed ID:
18852451
Buis JM, Broderick JB
Pyruvate formate-lyase activating enzyme: elucidation of a novel mechanism for glycyl radical formation
▸ Abstract
Pyruvate formate lyase activating enzyme is a member of a novel superfamily of enzymes that utilize S-adenosylmethionine to initiate radical catalysis. This enzyme has been isolated with several different iron-sulfur clusters, but single turnover monitored by EPR has identified the [4Fe-4S](1+) cluster as the catalytically active cluster; this cluster is believed to be oxidized to the [4Fe-4S](2+) state during turnover. The [4Fe-4S] cluster is coordinated by a three-cysteine motif common to the radical/S-adenosylmethionine superfamily, suggesting the presence of a unique iron in the cluster. The unique iron site has been confirmed by Mossbauer and ENDOR spectroscopy experiments, which also provided the first evidence for direct coordination of S-adenosylmethionine to an iron-sulfur cluster, in this case the unique iron of the [4Fe-4S] cluster. Coordination to the unique iron anchors the S-adenosylmethionine in the active site, and allows for a close association between the sulfonium of S-adenosylmethionine and the cluster as observed by ENDOR spectroscopy. The evidence to date leads to a mechanistic proposal involving inner-sphere electron transfer from the cluster to the sulfonium of S-adenosylmethionine, followed by or concomitant with C-S bond homolysis to produce a 5'-deoxyadenosyl radical; this transient radical abstracts a hydrogen atom from G734 to activate pyruvate formate lyase.
Pyruvate formate-lyase, evidence for an open conformation favored in the presence of its activating enzyme
▸ Abstract
Pyruvate formate-lyase-activating enzyme (PFL-AE) activates pyruvate formate-lyase (PFL) by generating a catalytically essential radical on Gly-734 of PFL. Crystal structures of unactivated PFL reveal that Gly-734 is buried 8 A from the surface of the protein in what we refer to here as the closed conformation of PFL. We provide here the first experimental evidence for an alternate open conformation of PFL in which: (i) the glycyl radical is significantly less stable; (ii) the activated enzyme exhibits lower catalytic activity; (iii) the glycyl radical undergoes less H/D exchange with solvent; and (iv) the T(m) of the protein is decreased. The evidence suggests that in the open conformation of PFL, the Gly-734 residue is located not in its buried position in the enzyme active site but rather in a more solvent-exposed location. Further, we find that the presence of the PFL-AE increases the proportion of PFL in the open conformation; this observation supports the idea that PFL-AE accesses Gly-734 for direct hydrogen atom abstraction by binding to the Gly-734 loop in the open conformation, thereby shifting the closed <--> open equilibrium of PFL to the right. Together, our results lead to a model in which PFL can exist in either a closed conformation, with Gly-734 buried in the active site of PFL and harboring a stable glycyl radical, or an open conformation, with Gly-734 more solvent-exposed and accessible to the PFL-AE active site. The equilibrium between these two conformations of PFL is modulated by the interaction with PFL-AE.
Pyruvate formate-lyase and its activation by pyruvate formate-lyase activating enzyme
▸ Abstract
The activation of pyruvate formate-lyase (PFL) by pyruvate formate-lyase activating enzyme (PFL-AE) involves formation of a specific glycyl radical on PFL by the PFL-AE in a reaction requiring S-adenosylmethionine (AdoMet). Surface plasmon resonance experiments were performed under anaerobic conditions on the oxygen-sensitive PFL-AE to determine the kinetics and equilibrium constant for its interaction with PFL. These experiments show that the interaction is very slow and rate-limited by large conformational changes. A novel AdoMet binding assay was used to accurately determine the equilibrium constants for AdoMet binding to PFL-AE alone and in complex with PFL. The PFL-AE bound AdoMet with the same affinity (∼6 μM) regardless of the presence or absence of PFL. Activation of PFL in the presence of its substrate pyruvate or the analog oxamate resulted in stoichiometric conversion of the [4Fe-4S](1+) cluster to the glycyl radical on PFL; however, 3.7-fold less activation was achieved in the absence of these small molecules, demonstrating that pyruvate or oxamate are required for optimal activation. Finally, in vivo concentrations of the entire PFL system were calculated to estimate the amount of bound protein in the cell. PFL, PFL-AE, and AdoMet are essentially fully bound in vivo, whereas electron donor proteins are partially bound.
Horitani M, Shisler K, Broderick WE, Hutcheson RU, Duschene KS, Marts AR, Hoffman BM, Broderick JB
Radical SAM catalysis via an organometallic intermediate with an Fe-[5'-C]-deoxyadenosyl bond
▸ Abstract
Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to cleave SAM to initiate diverse radical reactions. These reactions are thought to involve the 5'-deoxyadenosyl radical intermediate, which has not yet been detected. We used rapid freeze-quenching to trap a catalytically competent intermediate in the reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme. Characterization of the intermediate by electron paramagnetic resonance and (13)C, (57)Fe electron nuclear double-resonance spectroscopies reveals that it contains an organometallic center in which the 5' carbon of a SAM-derived deoxyadenosyl moiety forms a bond with the unique iron site of the [4Fe-4S] cluster. Discovery of this intermediate extends the list of enzymatic bioorganometallic centers to the radical SAM enzymes, the largest enzyme superfamily known, and reveals intriguing parallels to B12 radical enzymes.
