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.     

Cytosolic enzymes with a mitochondrial ancestry from the anaerobic chytrid Piromyces sp. E2

The anaerobic chytrid Piromyces sp. E2 lacks mitochondria, but contains hydrogen-producing organelles, the hydrogenosomes. We are interested in how the adaptation to anaerobiosis influenced enzyme compartmentalization in this organism. Random sequencing of a cDNA library from Piromyces sp. E2 resulted in the isolation of cDNAs encoding malate dehydrogenase, aconitase and acetohydroxyacid reductoisomerase. Phylogenetic analysis of the deduced amino acid sequences revealed that they are closely related to their mitochondrial homologues from aerobic eukaryotes. However, the deduced sequences lack N-terminal extensions, which function as mitochondrial leader sequences in the corresponding mitochondrial enzymes from aerobic eukaryotes. Subcellular fractionation and enzyme assays confirmed that the corresponding enzymes are located in the cytosol. As anaerobic chytrids evolved from aerobic, mitochondria-bearing ancestors, we suggest that, in the course of the adaptation from an aerobic to an anaerobic lifestyle, mitochondrial enzymes were retargeted to the cytosol with the concomitant loss of their N-terminal leader sequences.     

Multiple origins of hydrogenosomes: functional and phylogenetic evidence from the ADP/ATP carrier of the anaerobic chytrid Neocallimastix sp

A mitochondrial-type ADP/ATP carrier (AAC) has been identified in the hydrogenosomes of the anaerobic chytridiomycete fungus Neocallimastix sp. L2. Biochemical and immunocytochemical studies revealed that this ADP/ATP carrier is an integral component of hydrogenosomal membranes. Expression of the corresponding cDNA in Escherichia coli confers the ability on the bacterial host to incorporate ADP at significantly higher rates than ATP--similar to isolated mitochondria of yeast and animals. Phylogenetic analysis of this AAC gene (hdgaac) confirmed with high statistical support that the hydrogenosomal ADP/ATP carrier of Neocallimastix sp. L2 belongs to the family of veritable mitochondrial-type AACs. Hydrogenosome-bearing anaerobic ciliates possess clearly distinct mitochondrial-type AACs, whereas the potential hydrogenosomal carrier Hmp31 of the anaerobic flagellate Trichomonas vaginalis and its homologue from Trichomonas gallinae do not belong to this family of proteins. Also, phylogenetic analysis of genes encoding mitochondrial-type chaperonin 60 proteins (HSP 60) supports the conclusion that the hydrogenosomes of anaerobic chytrids and anaerobic ciliates had independent origins, although both of them arose from mitochondria.     

End-product induced metabolic shifts in <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> ATCC 27405

When attempting to increase yields of desirable end-products during fermentation, there is the possibility that increased concentrations of one product redirects metabolism towards the synthesis of less desired products. Changes in growth, final end-product concentrations, and activities of enzymes involved in pyruvate catabolism and fermentative end-product formation were studied in Clostridium thermocellum in response to the addition of individual end-products (H₂, acetate, ethanol, formate, and lactate) to the growth medium. These were added to the growth medium at concentrations ten times greater than those found at the end of growth in cultures grown under carbon-limited conditions using cellobiose (1.1 g l⁻¹) as model soluble substrate. Although growth rate and final cell biomass decreased significantly with the addition of all end-products, addition of individual end-products had less pronounced effects on growth. Metabolic shifts, represented by changes in final end-product concentrations, were observed; H₂ and acetate yields increased in the presence of exogenous ethanol and lactate, while ethanol yields increased in the presence of exogenous hydrogen (H₂), acetate, and lactate. Late exponential phase enzyme activity data of enzymes involved in pyruvate catabolism and end-product formation revealed no changes in enzyme levels greater than 2-fold in response to the presence of any given end-product, with the exception of pyruvate:formate lyase (PFL), ferredoxin-dependent hydrogenase (Fd-H₂ase), and pyruvate:ferredoxin oxidoreductase (PFO): PFL and Fd-H₂ase activities increased 2-fold in the presence of ethanol, while PFO activity decreased by 57% in the presence of sodium formate. Changes in enzyme levels did not necessarily correlate with changes in final end-product yields, suggesting that changes in final end-product yields may be governed by thermodynamic considerations rather than levels of enzyme expressed under the conditions tested. We demonstrate that bacterial metabolism may be manipulated in order to selectively improve desired product yields.     

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.     

Description of two anaerobic fungal strains from the bovine rumen and influence of diet on the fungal population in vivo

Neocallimastix sp. NC71 and Piromyces sp. PC12 isolated from the calf remen grew optimally at 39 degrees C and pH 6.5-6.7, utilized a wide range of mono-, oligo- and polysaccharides and exhibited CMCase, Avicelase, cellobiase, amylase and xylanase activities. The end-products of wheat straw fermentation by both strains were acetate, formate, ethanol and lactate. The number of Neocallimastix sp. zoospores in the rumen of cows in the first 3 h after feeding with hay-silage-concentrate diets varied from 7 x 10(3) to 5.4 x 10(5) ml-1; the number of uniflagellate zoospores varied from 10(4) to 10(5) ml-1. Fungal zoosporgenesis and colonization of plant substrates in the rumen were induced by feed intake and were favoured by increased levels of crude fibre in the diet.     

Dilution rate increases production of plant cell-wall degrading enzymes by anaerobic fungi in continuous-flow culture

The gut anaerobic fungi,Neocallimastix hurleyensis and aOrpinomyces sp., were grown in 100 mL batch and continuous-flow cultures on wheat straw at a concentration of 80 g dry matter/L of culture liquid. In batch cultures,N. hurleyensis and Orpinomyces sp. degraded only ca. 9% and 5% of the wheat straw, respectively. In continuous-flow cultures, however, the two fungi degraded 52-56% of the apparent dry matter of wheat straw. Both fungi were able to produce greater quantities (up to x 30) of cell-wall degrading enzymes (CMCase, xylanase, beta-glucosidase and beta-xylosidase) in continuous-flow cultures than in the corresponding batch cultures. Increasing the dilution rate in continuous-flow culture resulted in the production of increased enzyme activity for all the measured cell-wall degrading enzymes, with proportional relationships between dilution rate and the cumulative activities of beta-glucosidase and beta-xylosidase. Dilution rates, however, had no consistent effect on the cumulative production of the fermentation end-products, acetate, formate, D- and L-lactate from both fungi. In addition to acetate and formate,N. hurleyens is produced D- and L-lactate in both batch and continuous-flow cultures, whereas only trace amounts of L-lactate were detected in the Orpinomyces sp. cultures.     

Pyruvate formate lyase (PFL) and PFL activating enzyme in the chytrid fungus Neocallimastix frontalis: a free-radical enzyme system conserved across divergent eukaryotic lineages

Fermentative formate production involves the activity of pyruvate formate lyase, an oxygen-sensitive enzyme that employs a glycyl radical in its reaction mechanism. While common among anaerobic prokaryotes, this enzyme has so far been found in only two distantly related eukaryotic lineages, anaerobic chytridiomycetes and chlorophytes. Sequence comparisons of homologues from the chytridiomycetes Piromyces and Neocallimastix, the chlorophyte Chlamydomonas, and numerous prokaryotes suggest a single, eubacterial origin of eukaryotic pyruvate formate lyases. Pyruvate formate lyase activating enzyme introduces the glycyl radical into the pyruvate formate lyase protein chain. We discovered this enzyme, which had not previously been reported from eukaryotes, in the same two eukaryotic lineages and show that it shares a similar evolutionary history to pyruvate formate lyase. Sequences with high homology to pyruvate formate lyase activating enzyme were identified in the genomes of the anaerobic protozoan parasites Trichomonas vaginalis, Entamoeba histolytica, and Giardia intestinalis. While the occurrence of pyruvate formate lyase activating enzyme together with pyruvate formate lyase in fungi and chlorophytes was to be expected, the target protein of a glycyl radical enzyme-activating enzyme in these protozoa remains to be identified.     

Redirection of pyruvate catabolism in Lactococcus lactis by selection of mutants with additional growth requirements

Based on requirements for acetate or lipoic acid for aerobic (but not anaerobic) growth, Lactococcus lactis subsp. lactis mutants with impaired pyruvate catabolism were isolated following classical mutagenesis. Strains with defects in one or two of the enzymes, pyruvate formate-lyase (PFL), lactate dehydrogenase (LDH) and the pyruvate dehydrogenase complex (PDHC) were obtained. Growth and product formation of these strains were characterized. A PFL-defective strain (requiring acetate for anaerobic growth) displayed a two-fold increase in specific lactate production compared with the corresponding wild-type strain when grown anaerobically. LDH defective strains directed 91-96% of the pyruvate towards alpha-acetolactate, acetoin and diacetyl production when grown aerobically in the presence of acetate and absence of lipoic acid (a similar characteristic was observed in an LDH and PDHC defective strain in the presence of both acetate and lipoic acid) and more than 65% towards formate, acetate and ethanol production under anaerobic conditions. Another strain with defective PFL and LDH was strictly aerobic. However, a variant with strongly enhanced diacetyl reductase activities (NADH/NAD+ dependent diacetyl reductase, acetoin reductase and butanediol dehydrogenase activities) was selected from this strain under anaerobic conditions by supplementing the medium with acetoin. This strain is strictly aerobic, unless supplied with acetoin.     

Characterization of an anaerobic fungus from llama faeces.

An anaerobic fungus was isolated from llama faeces. Based on its morphological characteristics, polyflagellated zoospores, extensive rhizoid system and the formation of monocentric colonies, the fungus is assigned to the genus Neocallimastix. Neocallimastix sp. L2 is able to grow on several poly-, oligo- and monosaccharides. It differs from other Neocallimastix isolates in its inability to ferment inulin. Neocallimastix sp. L2 requires CO2 for growth. In the presence of 100% CO2 in the gas phase glucose is fermented to H2, CO2, formate, acetate, lactate, succinate and ethanol (33.8, 15.4, 74.3, 69.2, 26.7, 8.2, and 28.7 mmol per 100 mmol glucose, respectively). Reduced sulphur compounds can be used as sulphur source and ammonium or amino acids as nitrogen source. The temperature range for glucose fermentation is from 37 to 42 degrees C with an optimum of around 38 degrees C. The pH range for glucose fermentation is from pH 6 to pH 8 with a broad optimum between pH 6.5 and pH 7.5. The zoospores of Neocallimastix sp. L2 contain ribosomal 'globules' and hydrogenosomes. In the kinetosomes of the zoospores spurs, scoops and skirts are visible. In both the rhizoids and the sporangia 'crystal bodies' and hydrogenosomes are present. Mitochondria were not detected in either of these life stages.     

Influence of Metronidazole, CO, CO(2), and Methanogens on the Fermentative Metabolism of the Anaerobic Fungus Neocallimastix sp. Strain L2

The effects of metronidazole, CO, methanogens, and CO(2) on the fermentation of glucose by the anaerobic fungus Neocallimastix sp. strain L2 were investigated. Both metronidazole and CO caused a shift in the fermentation products from predominantly H(2), acetate, and formate to lactate as the major product and caused a lower glucose consumption rate and cell protein yield. An increased lactate dehydrogenase activity and a decreased hydrogenase activity were observed in cells grown under both culture conditions. In metronidazole-grown cells, the amount of hydrogenase protein was decreased compared with the amount in cells grown in the absence of metronidazole. When Neocallimastix sp. strain L2 was cocultured with the methanogenic bacterium Methanobrevibacter smithii, the fermentation pattern changed in the opposite direction: H(2) and acetate production increased at the expense of the electron sink products lactate, succinate, and ethanol. A concomitant decrease in the enzyme activities leading to these electron sink products was observed, as well as an increase in the glucose consumption rate and cell protein yield, compared with those of pure cultures of the fungus. Low levels of CO(2) in the gas phase resulted in increased H(2) and lactate formation and decreased production of formate, acetate, succinate, and ethanol, a decreased glucose consumption rate and cell protein yield, and a decrease in most of the hydrogenosomal enzyme activities. None of the tested culture conditions resulted in changed quantities of hydrogenosomal proteins. The results indicate that manipulation of the pattern of fermentation in Neocallimastix sp. strain L2 results in changes in enzyme activities but not in the proliferation or disappearance of hydrogenosomes.     

Molecular characteristics and transcription of the gene encoding a multifunctional alcohol dehydrogenase in relation to the deactivation of pyruvate formate-lyase in the ruminal bacterium<Emphasis Type="Italic"> Streptococcus bovis</Emphasis>

To clarify the deactivation mechanism of pyruvate formate-lyase (PFL) and its role in the regulation of fermentation in Streptococcus bovis, the molecular properties and genetic expression of multifunctional alcohol dehydrogenase (ADHE) were investigated. S. bovis was found to have ADHE, which was deduced to consist of 872 amino acids with a molecular mass of 97.4 kDa. The ADHE was shown to harbor three enzyme activities: (1) alcohol dehydrogenase, (2) coenzyme-A-linked acetaldehyde dehydrogenase that catalyzes the conversion of acetyl-CoA to ethanol, and (3) PFL deactivase. Similar to Escherichia coli ADHE, S. bovis ADHE required Fe2+ for its activity. The gene encoding ADHE ( adhE) was shown to be monocistronic. The level of adhE mRNA changed in parallel with the mRNA levels of the genes encoding PFL (pfl) and PFL-activating enzyme (act) as the growth conditions changed, although these genes are independently transcribed. Synthesis of ADHE, PFL-activating enzyme, and PFL appears to be regulated concomitantly. Overexpression of ADHE did not cause a change in the formate-to-lactate ratio. It is conceivable that ADHE is not significantly involved in the reversible inactivation of active PFL under anoxic conditions. Partition of the flow from pyruvate appears to be mainly regulated by the activities of lactate dehydrogenase and PFL.     

Pyruvate degradation via pyruvate formate-lyase (EC 2.3.1.54) and the enzymes of formate fermentation in the green alga Chlorogonium elongatum

Formate was formed in extracts of Chlorogonium elongatum via direct cleavage of pyruvate by a pyruvate formate-lyase (PFL, EC 2.3.1.54). The conversion of PFL to the catalytically active form required S-adenosylmethionine, ferric (2+), photoreduced deazariboflavin as reductant, pyruvate as allosteric effector and strict anaerobic conditions. At the optimum pH (pH 8.0), PFL catalyzed formate formation, pyruvate synthesis and the isotope exchange from [¹⁴C]formate into pyruvate with rates of 30.0, 1.5 and 1.2 nmol min^-1 mg^-1 protein, respectively. Treatment of the active enzyme with O₂ irreversibly inactivated PFL activity (half-time 2 min). In addition to PFL, the activities of phosphotransacetylase (EC 2.3.1.8), acetate kinase (EC 2.7.2.1), aldehyde dehydrogenase (CoA acetylating, EC 1.2.1.10) and alcohol dehydrogenase (EC 1.1.1.1) were also detected in extracts of C. elongatum. The occurrence of these enzymes indicates pyruvate degradation via a formate-fermentation pathway during anaerobiosis of algal cells in the dark.Abbreviations DTT dithiothreitol - Hepes 4-(2-hydroxyethyl)-1-piperazine+ethane sulfonic acid - PFL pyruvate formate-lyase     

过量表达苹果酸酶对E.coli FMJ39厌氧混合酸发酵的影响

为了考察苹果酸酶对厌氧混合酸发酵影响,从E.coli DH5α中PCR扩增苹果酸酶（NAD+-dependent, E.C1.1.1.38）基因sfcA,插入质粒pTrc99a构建了表达质粒pTrc99a-sfcA,有氧和厌氧的条件下,IPTG诱导在E.coli FMJ39（ldh,pfl）中均获得大量表达,从而构建和加强了一条在厌氧混合酸发酵中微弱的代谢途径。厌氧发酵结果表明,过量表达苹果酸酶会影响混合酸发酵中甲酸、乙酸、丁二酸途径。重组FMJ39甲酸和乙酸的量分别比FMJ39提高了17.58%和15.27%,丁二酸的量降低了26.87%,柠檬酸的量变化不大。证实即使pfl基因缺陷,高浓度的L-Thr和L-Ser也会诱导Tdc 操纵元把丙酮酸转化为甲酸和乙酸。实验结果为进一步改造和利用FMJ39奠定了基础。     

Effect of inactivation of nuo and ackA-pta on redistribution of metabolic fluxes in Escherichia coli.

The nuoA-N gene cluster encodes a transmembrane NADH:ubiquinone oxidoreductase (NDH-I) responsible for coupling redox chemistry to proton-motive force generation. Interactions between nuo and the acetate-producing pathway encoded by ackA-pta were investigated by examining the metabolic patterns of several mutant strains under anaerobic growth conditions. In an ackA-pta strain, the flux to acetate was decreased dramatically, whereas flux to lactate was increased significantly when compared with its parent strain; the fluxes to pyruvate and ethanol also increased slightly. In addition, pyruvate was excreted. A strain carrying the nuo mutation showed metabolic flux distribution similar to the wild type. The ackA-pta-nuo strain showed a different metabolic pattern. It not only exhibited reduced acetate accumulation but also significantly lower ethanol and formate synthesis. Metabolic flux distribution analysis suggests that the excessive carbon flux was redirected at the pyruvate node through the lactate dehydrogenase pathway for lactate formation rather than the pyruvate formate-lyase (PFL) pathway for acetyl-CoA and formate production. The diminished capacity through the formate and ethanol (ADH) pathways was not the result of genetic disruption of functional PFL or ADH production. The introduction of a Bacillus subtilis acetolactate synthase gene returned formate, ethanol, and lactate levels to those of the wild type (ackA(+)pta(+)nuo(+)) strain. Furthermore, transfer of a lactate dehydrogenase mutation yielded a strain producing ethanol as the sole fermentation product. As confirmation of the nuo effect, cultures of the ackA-pta strain, supplemented with an NDH-I inhibitor, produced intermediary levels of flux to ethanol and formate. Mutations in both ackA-pta and nuo are required to significantly reduce the flux through the PFL pathway.     

Hydrolytic activities of anaerobic fungi from wild blue bull (Boselaphus tragocamelus)

The anaerobic fungi play an active role in the plant fibre degradation by producing a wide array of potential hydrolytic enzymes in the rumen. In present study, 12 anaerobic fungal strains were isolated from the faecal samples of wild blue bull, and identified as species of Piromyces, Anaeromyces, Orpinomyces and Neocallimastix based on their morphological characteristics. Isolate WNG-12 (Piromyces sp.), showed maximum filter paper cellulase (23 mIU ml(-1)) and xylanase (127 mIU ml(-1)) activity, while WNG-5 (Piromyces sp.) showed maximum carboxymethyl cellulase activity (231 mIU ml(-1)). Based on the results obtained, it can be stated that Piromyces sp. WNG-12 may be a promising isolate in utilizing fibre rich diets in the rumen as evidenced by the production of hydrolytic enzymes in vitro.     

Characterization of hydrogenosomes and their role in glucose metabolism of Neocallimastix sp. L2

In the anaerobic fungus Neocallimastix sp. L2 fermentation of glucose proceeds via the Embden-Meyerhof-Parnas pathway. Enzyme activities leading to the formation of succinate, lactate, ethanol, and formate are associated with the cytoplasmic fraction. The enzymes malic enzyme, NAD(P)H: ferredoxin oxidoreductase, pyruvate: ferredoxin oxidoreductase, hydrogenase, acetate: succinate CoA transferase and succinate thiokinase leading to the formation of H₂, CO₂, acetate, and ATP are localized in microbodies. Thus, these organelles are identified as hydrogenosomes. In addition, the microbodies contain the O₂-scavenging enzymes NADH- and NADPH oxidase, while NAD(P)H peroxidase, catalase, or superoxide dismutase could not be detected. In cell-free extracts from zoospores of Neocallimastix sp. L2 the specific activities of hydrogenosomal enzymes as well as the quantities of these proteins are 2- to 6-fold higher than in mycelium extracts. These findings suggest that hydrogenosomes perform an important role-especially in zoospores — as H₂-evolving, ATP-generating and O₂-scavenging organelles.Abbrevations DTT Dithiotreitol - PEP Phosphoenol pyruvate     

In vitro degradation of wheat straw by anaerobic fungi from small ruminants

Anaerobic ruminal fungi may play an active role in fibre degradation as evidenced by the production of different fibrolytic enzymes in culture filtrate. In the present study, 16 anaerobic fungal strains were isolated from ruminal and faecal samples of sheep and goats. Based on their morphological characteristics they were identified as species of Anaeromyces, Orpinomyces, Piromyces and Neocallimastix. Isolated Neocallimastix sp. from goat rumen showed a maximum activity of CMCase (47.9 mIU ml(-1)) and filter paper cellulase (48.3 mIU ml(-1)), while Anaeromyces sp. from sheep rumen showed a maximum xylanolytic activity (48.3 mIU ml(-1)). The cellobiase activity for all the isolates ranged from 178.0-182.7 mIU ml(-1). Based on the enzymatic activities, isolated Anaeromyces sp. from sheep rumen and Neocallimastix sp. from goat rumen were selected for their potential of in vitro fibre degradation. The highest in vitro digestibility of NDF (23.2%) and DM (34.4%) was shown for Neocallimastix sp. from goat rumen, as compared to the digestibility of NDF and DM in the control group of 17.5 and 25.0%, respectively.     

Pyruvate formate lyase is structurally homologous to type I ribonucleotide reductase.

BACKGROUND: Pyruvate formate lyase (PFL) catalyses a key step in Escherichia coli anaerobic glycolysis by converting pyruvate and CoA to formate and acetylCoA. The PFL mechanism involves an unusual radical cleavage of pyruvate, involving an essential C alpha radical of Gly734 and two cysteine residues, Cys418 and Cys419, which may form thiyl radicals required for catalysis. We undertook this study to understand the structural basis for catalysis. RESULTS: The first structure of a fragment of PFL (residues 1-624) at 2.8 A resolution shows an unusual barrel-like structure, with a catalytic beta finger carrying Cys418 and Cys419 inserted into the centre of the barrel. Several residues near the active-site cysteines can be ascribed roles in the catalytic mechanism: Arg176 and Arg435 are positioned near Cys419 and may bind pyruvate/formate and Trp333 partially buries Cys418. Both cysteine residues are accessible to each other owing to their cis relationship at the tip of the beta finger. Finally, two clefts that may serve as binding sites for CoA and pyruvate have been identified. CONCLUSIONS: PFL has striking structural homology to the aerobic ribonucleotide reductase (RNR): the superposition of PFL and RNR includes eight of the ten strands in the unusual RNR alpha/beta barrel as well as the beta finger, which carries key catalytic residues in both enzymes. This provides the first structural proof that RNRs and PFLs are related by divergent evolution from a common ancestor.