Structure and mechanism of the glycyl radical enzyme pyruvate formate-lyase.

Pyruvate formate-lyase (PFL) from Escherichia coli uses a radical mechanism to reversibly cleave the C1-C2 bond of pyruvate using the Gly 734 radical and two cysteine residues (Cys 418, Cys 419). We have determined by X-ray crystallography the structures of PFL (non-radical form), its complex with the substrate analog oxamate, and the C418A,C419A double mutant. The atomic model (a dimer of 759-residue monomers) comprises a 10-stranded beta/alpha barrel assembled in an antiparallel manner from two parallel five-stranded beta-sheets; this architecture resembles that of ribonucleotide reductases. Gly 734 and Cys 419, positioned at the tips of opposing hairpin loops, meet in the apolar barrel center (Calpha-Sgamma = 3.7 A). Oxamate fits into a compact pocket where C2 is juxtaposed with Cys 418Sgamma (3.3 A), which in turn is close to Cys 419Sgamma (3.7 A). Our model of the active site is suggestive of a snapshot of the catalytic cycle, when the pyruvate-carbonyl awaits attack by the Cys 418 thiyl radical. We propose a homolytic radical mechanism for PFL that involves Cys 418 and Cys 419 both as thiyl radicals, with distinct chemical functions.     

Pyruvate formate-lyase activating enzyme: elucidation of a novel mechanism for glycyl radical formation

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.     

YfiD of Escherichia coli and Y06I of bacteriophage T4 as autonomous glycyl radical cofactors reconstituting the catalytic center of oxygen-fragmented pyruvate formate-lyase.

Reaction of oxygen with the glycyl radical in pyruvate formate-lyase (PFL) leads to cleavage of the polypeptide backbone between N-Calpha of Gly734. A recombinant protein comprising the core of PFL (Ser1-Ser733) is shown here to associate with the YfiD protein (14 kDa) of Escherichia coli and likewise with the homologous T4 encoded Y06I protein, yielding upon reaction with PFL activase a heterooligomeric PFL enzyme that has full catalytic activity (35 U/nmol). Treatment of the activated complexes with oxygen led to cleavage of the 14 kDa proteins into 11 and 3 kDa polypeptides as expected for the localization of the putative glycyl radical at Gly102 (YfiD) or Gly95 (Y06I). For the isolated fragments from Y06I, mass spectrometric analysis (nanoESI-MS) determined a C-terminal serine carboxamide in the 11 kDa fragment, and a N-terminal oxalyl modification in the 3 kDa fragment. We speculate that YfiD in E. coli and other facultative anaerobic bacteria has evolved as a "spare part" for PFL's glycyl radical domain, utilized for rapid recovery of PFL activity (and thus ATP generation) in cells that have experienced oxidative stress.     

A hydrogenosome with pyruvate formate-lyase: anaerobic chytrid fungi use an alternative route for pyruvate catabolism

The chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possess hydrogenosomes. Hydrogenosomes are highly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the first time that chytrid hydrogenosomes use pyruvate formate-lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytrid PFLs are encoded by a multigene family and are abundantly expressed in Piromyces sp. E2 and Neocallimastix sp. L2. Western blotting after cellular fractionation, proteinase K protection assays and determinations of enzyme activities reveal that PFL is present in the hydrogenosomes of Piromyces sp. E2. The main route of the hydrogenosomal carbon metabolism involves PFL; the formation of equimolar amounts of formate and acetate by isolated hydrogenosomes excludes a significant contribution by PFO. Our data support the assumption that chytrid hydrogenosomes are unique and argue for a polyphyletic origin of these organelles.     

Modification of Cys-418 of pyruvate formate-lyase by methacrylic acid, based on its radical mechanism

The recently determined crystal structure of pyruvate formate-lyase (PFL) suggested a new view of the mechanism of this glycyl radical enzyme, namely that intermediary thiyl radicals of Cys-418 and Cys-419 participate in different ways [Becker, A. et al. (1999) Nat. Struct. Biol. 6, 969-975]. We report here a suicide reaction of PFL that occurs with the substrate-analog methacrylate with retention of the protein radical (K(I)=0.42 mM, k(i)=0.14 min(-1)). Using [1-(14)C]methacrylate (synthesized via acetone cyanhydrin), the reaction end-product was identified by peptide mapping and cocrystallization experiments as S-(2-carboxy-(2S)-propyl) substituted Cys-418. The stereoselectivity of the observed Michael addition reaction is compatible with a radical mechanism that involves Cys-418 thiyl as nucleophile and Cys-419 as H-atom donor, thus supporting the functional assignments of these catalytic amino acid residues derived from the protein structure.     

X-ray structure of pyruvate formate-lyase in complex with pyruvate and CoA. How the enzyme uses the Cys-418 thiyl radical for pyruvate cleavage

The glycyl radical enzyme pyruvate formate-lyase (PFL) synthesizes acetyl-CoA and formate from pyruvate and CoA. With the crystal structure of the non-radical form of PFL in complex with its two substrates, we have trapped the moment prior to pyruvate cleavage. The structure reveals how the active site aligns the scissile bond of pyruvate for radical attack, prevents non-radical side reactions of the pyruvate, and confines radical migration. The structure shows CoA in a syn conformation awaiting pyruvate cleavage. By changing to an anti conformation, without affecting the adenine binding mode of CoA, the thiol of CoA could pick up the acetyl group resulting from pyruvate cleavage.     

Ultrastructure and pyruvate formate-lyase radical quenching property of the multienzymic AdhE protein of Escherichia coli.

The AdhE protein of Escherichia coli is a homopolymer of 96-kDa subunits harboring three Fe(2+)-dependent catalytic functions: acetaldehyde-CoA dehydrogenase, alcohol dehydrogenase, and pyruvate formatelyase (PFL) deactivase. By negative staining electron microscopy, we determined a helical assembly of 20-60 subunits into rods of 45-120 nm in length. The subunit packing is widened along the helix axis when Fe2+ and NAD are present. Chymotrypsin dissects the AdhE polypeptide between Phe762 and Ser763, thereby retaining the alcohol dehydrogenase activity on the NH2-terminal core, but destroying all other activities. PFL deactivation, i.e. quenching of the glycyl radical in PFL by the AdhE protein, was examined with respect to cofactor involvements (Fe2+, NAD, and CoA). This process is coupled to NAD reduction and requires the intact CoA sulfhydryl group. Pyruvate and NADH are inhibitors that affect the steady-state level of the radical form of PFL in a reconstituted interconversion cycle. Studies of cell cultures found that PFL deactivation in situ is initiated at redox potentials of greater than or equal to +100 mV. Our results provide insights into the structure/function organization of the AdhE multienzyme and give a rationale for how its PFL radical quenching activity may be suppressed in situ to enable effective glucose fermentation.     

Role of pyruvate and S-adenosylmethioine in activating the pyruvate formate-lyase of Escherichia coli

The pyruvate formate-lyase activity of extracts of Escherichia coli is stimulated and the dilution effect is abolished by the addition of pyruvate to the extract. The activity can be purified fourfold from pyruvate-supplemented extracts by isoelectric precipitation under anaerobic conditions. The activity of extracts not supplemented with pyruvate has been separated into two fractions by treatment with protamine sulfate-fraction PS, the soluble portion, and fraction N, an extract of the precipitate formed upon the addition of protamine sulfate. After treatment of these fractions with charcoal, pyruvate formate-lyase activity is stimulated by the addition of S-adenosylmethionine. When sodium pyruvate is added to the crude extract before the fractionation, fraction PS has full enzymatic activity and is not stimulated by fraction N or by S-adenosylmethionine. Incubation of the inactive fractions with pyruvate and S-adenosylmethionine in the absence of other substrates similarly results in a highly active preparation, not subject to the "dilution effect" obtained when the fractions are added separately to the assay. These observations suggest that the component in the protamine supernatant fraction is activated by the other fraction and that S-adenosylmethionine and pyruvate are required for the activation reaction. The activating factor present in the protamine precipitate fraction may be further purified by heating for 10 min at 100 C under H(2) atmosphere. The yield of this factor from crude extract is not affected by activation of the pyruvate formate-lyase of the extract, indicating that the factor acts catalytically. The requirement for pyruvate is only partially satisfied by alpha-ketobutyrate and not at all by other alpha-keto acids, acetyl phosphate, or adenosine triphosphate. The rate of activation is maximal at 0.01 m sodium pyruvate and 3 x 10(-4)mS-adenosylmethionine; it is linearly dependent on the amount of activating factor added. The rate of activation is the same when the activation reaction is initiated by addition of any of the four required components, indicating that no slow step of activation can be carried out by any three of the components. A similar pyruvate formate-lyase system was found in extracts of the methionine/B(12) autotroph 113-3, grown with methionine supplement, indicating that vitamin B(12) derivatives do not participate in the system.     

A novel reaction of S-adenosyl-L-methionine correlated with the activation of pyruvate formate-lyase

Conversion of the inactive form of pyruvate formate-lyase to the catalytically active enzyme is accomplished by the Fe-dependent ‘enzyme II’; reduced flavodoxin, S-adenosyl-L-methionine and the effector pyruvate are required. It was found that adenosylmethionine is reductively processed during activation of pyruvate formate-lyase to yield methionine, adenine and 5-deoxyribose. We suggest that transient adenosylation of enzyme II is required for its function as a converter enzyme.     

Synthesis and posttranslational regulation of pyruvate formate-lyase in Lactococcus lactis

The enzyme pyruvate formate-lyase (PFL) from Lactococcus lactis was produced in Escherichia coli and purified to obtain anti-PFL antibodies that were shown to be specific for L. lactis PFL. It was demonstrated that activated L. lactis PFL was sensitive to oxygen, as in E. coli, resulting in the cleavage of the PFL polypeptide. The PFL protein level and its in vivo activity and regulation were shown by Western blotting, enzyme-linked immunosorbent assay, and metabolite measurement to be dependent on the growth conditions. The PFL level during anaerobic growth on the slowly fermentable sugar galactose was higher than that on glucose. This shows that variation in the PFL protein level may play an important role in the regulation of metabolic shift from homolactic to mixed-acid product formation, observed during growth on glucose and galactose, respectively. During anaerobic growth in defined medium, complete activation of PFL was observed. Strikingly, although no formate was produced during aerobic growth of L. lactis, PFL protein was indeed detected under these conditions, in which the enzyme is dispensable due to the irreversible inactivation of PFL by oxygen. In contrast, no oxygenolytic cleavage was detected during aerobic growth in complex medium. This observation may be the result of either an effective PFL deactivase activity or the lack of PFL activation. In E. coli, the PFL deactivase activity resides in the multifunctional alcohol dehydrogenase ADHE. It was shown that in L. lactis, ADHE does not participate in the protection of PFL against oxygen under the conditions analyzed. Our results provide evidence for major differences in the mechanisms of posttranslational regulation of PFL activity in E. coli and L. lactis.     

Pyruvate formate-lyase (inactive form) and pyruvate formate-lyase activating enzyme of Escherichia coli: Isolation and structural properties

The catalytically active form (E_a) of pyruvate formate-lyase in Escherichia coli cells is generated from an inactive form of the enzyme (E_i) through a post-translational process that requires a distinct activating enzyme and is linked to the cleavage of adenosylmethionine to methionine and 5′-deoxyadenosine. E_i and the activating enzyme were purified to homogeneity and structurally characterized. E_i has an α₂ oligomeric structure (2 × 85 kDa) and contains no cofactor. The amino acid composition has been determined. Out of a total of six cysteinyl residues per subunit, one shows an unusually fast reaction with iodoacetate (k₂ = 7 (m^? s^?) at pH 6.8, 30 °C), which is accompanied by loss of the activatability of the enzyme. The 1500-fold purified activating enzyme is a monomeric protein of 30 kDa. It contains a covalently bound, as yet unidentified chromophoric factor which has an optical absorption peak at 388 nm. Further studies of the in situ state of pyruvate formate-lyase detected a reversible backconversion of the active form E_a into E_i when anaerobic cells become nutrient-depleted.     

Anaerobic regulation of pyruvate formate-lyase from Escherichia coli K-12.

The anaerobic regulation of the gene encoding pyruvate formate-lyase from Escherichia coli was investigated. Expression of a pfl'-'lacZ protein fusion demonstrated that the gene is subject to a 12-fold anaerobic induction which can be stimulated a further 2-fold by the addition of pyruvate to the growth medium. Construction of a strain deleted for pfl verified that either pyruvate or a metabolite of glycolysis functions as an inducer of pfl gene expression. Complete anaerobic induction required the presence of a functional fnr gene product. However, the dependence was not absolute since a two- to threefold anaerobic induction could still be observed in an fnr mutant. These results could be confirmed immunologically by analyzing the levels of pyruvate formate-lyase protein present in cells grown under various conditions. It was also shown that pfl'-'lacZ expression was partially repressed by nitrate and that this repression was mediated by the narL gene product.     

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

Escherichia 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 detectable, 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 beta-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.     

Expression of pyruvate formate-lyase of Escherichia coli from the cloned structural gene

It is shown here that a plasmid (p29) derived from the transducing phage aspC2 (Christiansen and Pedersen 1981) codes for pyruvate formate-lyase. The identity of the 80 kilodaltons (kd) gene product of plasmid p29 with the pyruvate formate-lyase polypeptide was proven (i) by comigration of the gene product expressed in the maxicell system with purified enzyme on O'Farrell gels, and (ii) by comparison of the peptide maps obtained from limited proteolysis. In vivo the 80 kd form of the enzyme was proteolytically converted to a 78 kd polypeptide. The two polypeptides (80 kd and 78 kd) and their charge isomers present in purified enzyme preparations are therefore products of a single gene.Aerobically grown cells of Escherichia coli contained a basal level of pyruvate formate-lyase which was derepressed 5-to 10-fold under anaerobiosis. Derepression also occurred during anaerobic growth on glycerol plus fumarate. Presence of plasmid p29 caused overproduction of pyruvate formatelyase, 11-fold upon anaerobic growth on glucose, 14-fold upon aerobic growth on glucose and 33-fold upon aerobic growth at the expense of D-lactate.Non-Standard Abbreviation MOPS 4-morpholine-propane sulfonic     

The free radical of pyruvate formate-lyase. Characterization by EPR spectroscopy and involvement in catalysis as studied with the substrate-analogue hypophosphite

The first-derivative EPR spectrum of the active form of Escherichia coli pyruvate formate-lyase shows an asymmetric doublet with partially resolved hyperfine splittings (g = 2.0037). Isotope substitution studies demonstrated couplings of a carbon-centered unpaired electron to a solvent-exchangeable proton (a = 1.5 mT) and to further hydrogen nuclei (a = 0.36 and 0.57 mT). By selective incorporation of unlabelled tyrosine into 2H-labelled enzyme protein, a tyrosyl radical structure has been ruled out. Circumstantial evidence indicates that the organic free radical, which also displays an ultraviolet absorption signal at 365 nm, is located on a standard amino acid residue of the polypeptide chain. EPR signal quantification found a stoichiometry of 1 spin per active site. The formate analogue hypophosphite has been characterized as a specific kcat inhibitor of pyruvate formate-lyase which destroys the enzyme radical. Protein-linked 1-hydroxyethylphosphonate was previously described as the dead-end product after reaction of the analogue with the intermediary acetyl-enzyme form of the catalytic cycle [W. Plaga et al. (1988) Eur. J. Biochem. 178, 445-450]. EPR spectroscopy of this system has now identified the corresponding alpha-phosphoryl radical as a reaction intermediate [g = 2.0032; a(P) = 2.72 mT, a(3H) = 1.96 mT]; it showed a half-life of about 20 min at 0 degrees C. This finding proves that the enzyme radical is a hydrogen-atom-transferring coenzymic element.     

Rescuing activity of oxygen-damaged pyruvate formate-lyase by a spare part protein

Pyruvate formate-lyase (PFL) is a glycyl radical enzyme (GRE) that converts pyruvate and coenzyme A into acetyl-CoA and formate in a reaction that is crucial to the primary metabolism of many anaerobic bacteria. The glycyl radical cofactor, which is posttranslationally installed by a radical S-adenosyl-L-methionine (SAM) activase, is a simple and effective catalyst, but is also susceptible to oxidative damage in microaerobic environments. Such damage occurs at the glycyl radical cofactor, resulting in cleaved PFL (cPFL). Bacteria have evolved a spare part protein termed YfiD that can be used to repair cPFL. Previously, we obtained a structure of YfiD by NMR spectroscopy and found that the N-terminus of YfiD was disordered and that the C-terminus of YfiD duplicates the structure of the C-terminus of PFL, including a β-strand that is not removed by the oxygen-induced cleavage. We also showed that cPFL is highly susceptible to proteolysis, suggesting that YfiD rescue of cPFL competes with protein degradation. Here, we probe the mechanism by which YfiD can bind and restore activity to cPFL through enzymatic and spectroscopic studies. Our data show that the disordered N-terminal region of YfiD is important for YfiD glycyl radical installation but not for catalysis, and that the duplicate β-strand does not need to be cleaved from cPFL for YfiD to bind. In fact, truncation of this PFL region prevents YfiD rescue. Collectively our data suggest the molecular mechanisms by which YfiD activation is precluded both when PFL is not damaged and when it is highly damaged.