Involvement of pyruvate dehydrogenase in product formation in pyruvate-limited anaerobic chemostat cultures of Enterococcus faecalis NCTC 775

Enterococcus faecalis NCTC 775 was grown anaerobically in chemostat culture with pyruvate as the energy source. At low culture pH values, high in vivo and in vitro activities were found for both pyruvate dehydrogenase and lactate dehydrogenase. At high culture pH values the carbon flux was shifted towards pyruvate formate lyase. Some mechanisms possibly involved in this metabolic switch are discussed. In particular attention is paid to the NADH/NAD ratio (redox potential) and the fructose-1,6-bisphosphate-dependent lactate dehydrogenase activity as possible regulatory factors.Abbreviations PDH pyruvate dehydrogenase complex (EC 1.2.2.2) - PFL pyruvate formate lyase (EC 2.3.1.54) - LDH lactate dehydrogenase (EC 1.1.1.27) - FBP fructose-1,6-bisphosphate - MTT 3-(4,5-dimethyl-thiazoyl-2)-2,5-diphenyltetrazolium bromide - TPP thiamine pyrophosphate     

L-lactate dehydrogenase from leaves of Capsella bursa-pastoris (L.) Med.

By ammonium sulfate fractionation and gel filtration an enzyme preparation which catalyzed NAD⁺-dependent L-lactate oxidation (10^-4 kat kg^-1 protein), as well as NADH-dependent pyruvate reduction (10^-3 kat kg^-1 protein), was obtained from leaves of Capsella bursa-pastoris. This lactate dehydrogenase activity was not due to an unspecific activity of either glycolate oxidase, glycolate dehydrogenase, hydroxypyruvate reductase, alcohol dehydrogenase, or a malate oxidizing enzyme. These enzymes could be separated from the protein displaying lactate dehydrogenase activity by gel filtration and electrophoresis and distinguished from it by their known properties. The enzyme under consideration does not oxidize D-lactate, and reduces pyruvate to L-lactate (the configuration of which was determined using highly specific animal L-lactate dehydrogenase). Based on these results the studied Capsella leaf enzyme is classified as L-lactate dehydrogenase (EC 1.1.1.27). It has a K_m value of 0.25 mmol l^-1 (pH 7.0, 0.3 mmol l^-1 NADH) for pyruvate and of 13 mmol l^-1 (pH 7.8, 3 mmol l^-1 NAD⁺) for L-lactate. Lactate dehydrogenase activity was also detected in the leaves of several other plants.Abbreviation FMN flavin adenine mononucleotide     

Formation of formate and hydrogen, and flux of reducing equivalents and carbon in Ruminococcus flavefaciens Fd-1

A pathway for conversion of the metabolic intermediate phosphoenolpyruvate (PEP) and the formation of acetate, succinate, formate, and H₂ in the anaerobic cellulolytic bacterium Ruminococcus flavefaciens FD-1 was constructed on the basis of enzyme activities detected in extracts of cells grown in cellulose- or cellobiose-limited continuous culture. PEP was converted to acetate and CO₂ (via pyruvate kinase, pyruvate dehydrogenase, and acetate kinase) or carboxylated to form succinate (via PEP carboxykinase, malate dehydrogenase, fumarase, and fumarate reductase). Lactate was not formed even during rapid growth (batch culture, µ = 0.35/h). H₂ was formed by a hydrogenase rather than by cleavage of formate, and ¹³C-NMR and¹⁴ C-exchange reaction data indicated that formate was produced by CO₂ reduction, not by a cleavage of pyruvate. The distribution of PEP into the acetate and succinate pathways was not affected by changing extracellular pH and growth rates within the normal growth range. However, increasing growth rate from 0.017/h to 0.244/h resulted in a shift toward formate production, presumably at the presence of H₂. This shift suggested that reducing equivalents could be balanced through formate or H₂ production without affecting the yields of the major carbon-containing fermentation endproducts.     

Biochemical and physiological characterization of the pyruvate formate-lyase Pfl1 of Chlamydomonas reinhardtii, a typically bacterial enzyme in a eukaryotic alga

The unicellular green alga Chlamydomonas reinhardtii has a special type of anaerobic metabolism that is quite unusual for eukaryotes. It has two oxygen-sensitive [Fe-Fe] hydrogenases (EC 1.12.7.2) that are coupled to photosynthesis and, in addition, a formate- and ethanol-producing fermentative metabolism, which was proposed to be initiated by pyruvate formate-lyase (Pfl; EC 2.3.1.54). Pfl enzymes are commonly found in prokaryotes but only rarely in eukaryotes. Both the hydrogen- and the formate/ethanol-producing pathways are involved in a sustained anaerobic metabolism of the alga, which can be induced by sulfur depletion in illuminated cultures. Before now, the presence of a Pfl protein in C. reinhardtii was predicted from formate secretion and the homology of the deduced protein of the PFL1 gene model to known Pfl enzymes. In this study, we proved the formate-producing activity of the putative Pfl1 enzyme by heterologous expression of the C. reinhardtii PFL1 cDNA in Escherichia coli and subsequent in vitro activity tests of the purified protein. Furthermore, a Pfl-deficient E. coli strain secretes formate when expressing the PFL1 cDNA of C. reinhardtii. We also examined the Pfl1 fermentation pathway of C. reinhardtii under the physiological condition of sulfur depletion. Genetic and biochemical analyses show that sulfur-depleted algae express genes encoding enzymes acting downstream of Pfl1 and also potentially ethanol-producing enzymes, such as pyruvate decarboxylase (EC 4.1.1.1) or pyruvate ferredoxin oxidoreductase (EC 1.2.7.1). The latter enzymes might substitute for Pfl1 activity when Pfl1 is specifically inhibited by hypophosphite.     

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.     

Metabolic regulation of carbon and electron flow as a function of pH during growth of Sarcina ventriculi

The physiology and biochemistry of Sarcina ventriculi was studied in order to determine adaptations made by the organism to changes in environmental pH. The organism altered carbon and electron flow from acetate, formate and ethanol production at neutral pH, to predominantly ethanol production at pH 3.0. Increased levels of pyruvate dehydrogenase (relative to pyruvate decarboxylase) and acetaldehyde dehydrogenase occurred when the organism was grown at neutral pH, indicating the predominance of carbon flux through the oxidative branch of the pathway for pyruvate metabolism. When the organism was grown at acid pH, there was a significant increase in pyruvate decarboxylase levels and a decrease in acetaldehyde dehydrogenase, causing flux through the non-oxidative branch of the pathway. CO₂ reductase and formate dehydrogenase were not regulated as a function of growth pH. Pyruvate dehydrogenase possessed Michaelis-Menten kinetics for pyruvate with an apparent K _m of 2.5 mM, whereas pyruvate decarboxylase exhibited sigmoidal kinetics, with a S_0.5 of 12.0 mM. Differences in total protein banding patterns from cells grown at pH extremes suggested that synthesis of pyruvate decarboxylase and other enzymes was in part responsible for metabolic regulation of the fermentation products formed.     

Characterization of mannitol metabolism in the mangrove red algaCaloglossa leprieurii (Montagne) J.Agardh

A metabolic pathway, known as the mannitol cycle in fungi, has been identified as a new entity in the eulittoral mangrove red algaCaloglossa leprieurii (Montagne) J. Agardh. Three specific enzymes, mannitol-1-phosphate dehydrogenase (Mt1PDH; EC 1.1.1.17), mannitol-1-phosphatase (MtlPase; EC 3.1.3.22), mannitol dehydrogenase (MtDH; EC 1.1.1.67) and one nonspecific hexokinase (HK; EC 2.7.1.1) were determined and biochemically characterized in cell-free extracts. Mannitol-1-phosphate dehydrogenase showed activity maxima at pH 7.0 [fructose-6-phosphate (F6P) reduction] and pH 8.5 [oxidation of mannitol-1-phosphate (Mt1P)], and a very high specificity for both carbohydrate substrates. TheK _m values were 1.4 mM for F6P, 0.09 mM for MOP, 0.020 mM for NADH and 0.023 mM for NAD⁺. For the dephosphorylation of MOP, MtlPase exhibited a pH optimum at 7.2, aK _m value of 1.2 mM and a high requirement of Mg²⁺ for activation. Mannitol dehydrogenase had activity maxima at pH 7.0 (fructose reduction) and pH 9.8 (mannitol oxidation), and was less substrate-specific than Mt1PDH and MtlPase, i.e. it also catalyzed reactions in the oxidative direction with arabitol (64.9%), sorbitol (31%) and xylitol (24.8%). This enzyme showedK _m values of 39 mM for fructose, 7.9 mM for mannitol, 0.14 mM for NADH and 0.075 mM for NAD⁺. For the non-specific HK, only theK _m values for fructose (0.19 mM) and glucose (7.5 mM) were determined. The activities of the anabolic enzymes Mt1PDH and MtlPase were always at least two orders of magnitude higher than those of the degradative enzymes, indicating a net carbon flow towards a high intracellular mannitol pool. The function of mannitol metabolism inC. leprieurii as a biochemical adaptation to the environmental extremes in the mangrove habitat is discussed.Abbreviations F6P fructose-6-phosphate - HK hexokinase - Mt1P mannitol-1-phosphate - Mt1PDH mannitol-1-phosphate dehydrogenase - Mt1Pase mannitol-1-phosphatase - MtDH mannitol dehydrogenase     

d-Xylose catabolism in Bacteroides xylanolyticus X5-1

The xylose metabolism of Bacteroides xylanolyticus X5-1 was studied by determining specific enzyme activities in cell free extracts, by following ¹³C-label distribution patterns in growing cultures and by mass balance calculations. Enzyme activities of the pentose phosphate pathway and the Embden-Meyerhof-Parnas pathway were sufficiently high to account for in vivo xylose fermentation to pyruvate via a combination of these two pathways. Pyruvate was mainly oxidized to acetyl-CoA, CO₂ and a reduced cofactor (ferredoxin). Part of the pyruvate was converted to acetyl-CoA and formate by means of a pyruvate-formate lyase. Acetyl-CoA was either converted to acetate by a combined action of phosphotransacetylase and acetate kinase or reduced to ethanol by an acetaldehyde dehydrogenase and an ethanol dehydrogenase. The latter two enzymes displayed both a NADH- and a NADPH-linked activity. Cofactor regeneration proceeded via a reduction of intermediates of the metabolism (i.e. acetyl-CoA and acetaldehyde) and via proton reduction. According to the deduced pathway about 2.5 mol ATP are generated per mol of xylose degraded.Abbreviations PPP Pentose phosphate pathway - PKP phosphoketolase pathway     

Metabolism of One-Carbon Compounds by the Ruminal Acetogen Syntrophococcus sucromutans

Syntrophococcus sucromutans is the predominant species capable of O demethylation of methoxylated lignin monoaromatic derivatives in the rumen. The enzymatic characterization of this acetogen indicated that it uses the acetyl coenzyme A (Wood) pathway. Cell extracts possess all the enzymes of the tetrahydrofolate pathway, as well as carbon monoxide dehydrogenase, at levels similar to those of other acetogens using this pathway. However, formate dehydrogenase could not be detected in cell extracts, whether formate or a methoxyaromatic was used as electron acceptor for growth of the cells on cellobiose. Labeled bicarbonate, formate, [1-¹⁴C] pyruvate, and chemically synthesized O-[methyl-¹⁴C]vanillate were used to further investigate the catabolism of one-carbon (C₁) compounds by using washed-cell preparations. The results were consistent with little or no contribution of formate dehydrogenase and pointed out some unique features. Conversion of formate to CO₂ was detected, but labeled formate predominantly labeled the methyl group of acetate. Labeled CO₂ readily exchanged with the carboxyl group of pyruvate but not with formate, and both labeled CO₂ and pyruvate predominantly labeled the carboxyl group of acetate. No CO₂ was formed from O demethylation of vanillate, and the acetate produced was position labeled in the methyl group. The fermentation pattern and specific activities of products indicated a complete synthesis of acetate from pyruvate and the methoxyl group of vanillate.     

Pyruvate Formate-lyase and Its Activation by Pyruvate Formate-lyase Activating Enzyme

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.     

Growth phase-dependant enzyme profile of pyruvate catabolism and end-product formation in Clostridium thermocellum ATCC 27405

End-product synthesis and enzyme activities involved in pyruvate catabolism, H₂ synthesis, and ethanol production in mid-log (OD₆₀₀  0.25), early stationary (OD₆₀₀  0.5), and stationary phase (OD₆₀₀  0.7) cell extracts were determined in Clostridium thermocellum ATCC 27405 grown in batch cultures on cellobiose. Carbon dioxide, hydrogen, ethanol, acetate and formate were major end-products and their production paralleled growth and cellobiose consumption. Lactate dehydrogenase, pyruvate:formate lyase, pyruvate:ferredoxin oxidoreductase, methyl viologen-dependant hydrogenase, ferredoxin-dependant hydrogenase, NADH-dependant hydrogenase, NADPH-dependant hydrogenase, NADH-dependant acetaldehyde dehydrogenase, NADH-dependant alcohol dehydogenase, and NADPH-dependant alcohol dehydrogenase activities were detected in all extracts, while pyruate dehydrogenase and formate dehydrogenase activities were not detected. All hydrogenase activities decreased (2–12-fold) as growth progressed from early exponential to stationary phase. Alcohol dehydrogenase activities fluctuated only marginally (<45%), while lactate dehydrogenase, pyruvate:formate lyase, and pyruvate:ferredoxin oxidoreductase remained constant in all cell extracts. We have proposed a pathway involved in pyruvate catabolism and end-product formation based on enzyme activity profiles in conjunction with bioinformatics analysis.     

Acetate formation from CO and CO2 by cell extracts of Peptostreptococcus productus (strain Marburg)

Cell extracts of Peptostreptococcus productus (strain Marburg) obtained from CO grown cells mediated the synthesis of acetate from CO plus CO₂ at rates of 50 nmol/min × mg of cell protein. ¹⁴CO was specifically incorporated into C₁ of acetate. No label exchange occurred between ¹⁴C₁ of acetyl-CoA and CO, indicating that ¹⁴CO incorporation into acetate was by net synthesis rather than by an exchange reaction. In acetate synthesis from CO plus CO₂ the latter substrate could be replaced to some extent by formate or methyl tetrahydrofolate as the methyl donor. The methyl group of methyl cobalamin was incorporated into acetate ony at very low activities. The cell extracts contained high levels of enzyme activities involved in acetate or cell carbon synthesis from CO₂. The following enzymic activities were detected: CO: methyl viologen oxidoreductase, formate dehydrogenase, formyl tetrahydrofolate synthetase, methenyl tetrahydrofolate cyclohydrolase, methylene tetrahydrofolate dehydrogenase, methylene tetrahydrofolate reductase, phosphate acetyltransferase, acetate kinase, hydrogenase, NADPH: benzyl viologen oxidoreductase, and pyruvate synthase. Some kinetic and other properties were studied.     

Induction and regulation of ribulose bisphosphate carboxylase activity in Rhizobium japonicum during formate-dependent growth

Rhizobium japonicum CJ1 was capable of growing using formate as the sole source of carbon and energy. During aerobic growth on formate a cytoplasmic NAD⁺-dependent formate dehydrogenase and ribulose bisphosphate carboxylase activity was demonstrated in cell-free extracts, but hydrogenase enzyme activity could not be detected. Under microaerobic growth conditions either formate or hydrogen metabolism could separately or together support ribulose bisphosphate carboxylase-dependent CO₂ fixation. A number of R. japonicum strains defective in hydrogen uptake activity were shown to metabolise formate and induce ribulose bisphosphate carboxylase activity. The induction and regulation of ribulose bisphosphate carboxylase is discussed.Abbreviations hup hydrogen uptake - MOPS 3-(N-morpholino)-propanesulphonate - TSA tryptone soya agar - RuBP ribulose 1,5-bisphosphate - FDH formate dehydrogenase     

Energetics of mixotrophic and autotrophic C1-metabolism by Thiobacillus acidophilus

Although the facultatively autotrophic acidophile Thiobacillus acidophilus is unable to grow on formate and formaldehyde in batch cultures, cells from glucose-limited chemostat cultures exhibited substrate-dependent oxygen uptake with these C₁-compounds. Oxidation of formate and formaldehyde was uncoupler-sensitive, suggesting that active transport was involved in the metabolism of these compounds. Formate- and formaldehyde-dependent oxygen uptake was strongly inhibited at substrate concentrations above 150 and 400 M, respectively. However, autotrophic formate-limited chemostat cultures were obtained by carefully increasing the formate to glucose ratio in the reservoir medium of mixotrophic chemostat cultures. The molar growth yield on formate (Y=2.5 g ·mol^-1 at a dilution rate of 0.05 h^-1) and RuBPCase activities in cell-free extracts suggested that T. acidophilus employs the Calvin cycle for carbon assimilation during growth on formate. T. acidophilus was unable to utilize the C₁-compounds methanol and methylamine. Formate-dependent oxygen uptake was expressed constitutively under a variety of growth conditions. Cell-free extracts contained both dye-linked and NAD-dependent formate dehydrogenase activities. NAD-dependent oxidation of formaldehyde required reduced glutathione. In addition, cell-free extracts contained a dye-linked formaldehyde dehydrogenase activity. Mixotrophic growth yields were higher than the sum of the heterotrophic and autotrophic yields. A quantitative analysis of the mixotrophic growth studies revealed that formaldehyde was a more effective energy source than formate.     

Carbohydrate Metabolism of the Saccharolytic Alkaliphilic Anaerobes Halonatronum saccharophilum,Amphibacillus fermentum,and Amphibacillus tropicus

The saccharolytic anaerobic bacteria Halonatronum saccharophilum, Amphibacillus fermentum, and Amphibacillus tropicus produce formate, the main fermentation product. In the alkaliphilic community, formate is used as the preferential substrate for sulfate reduction. To reveal the pathways of carbohydrate fermentation by these bacteria, the activity of the key enzymes of carbohydrate metabolism and their pH dependence was studied. It was established that H. saccharophilum utilized glucose by the fructose bisphosphate and hexose monophosphate pathways, and A. tropicus, by the fructose bisphosphate and Entner–Doudoroff pathways. The activity of the key enzymes of all three pathways of glucose metabolism was detected in Amphibacillus fermentum. According to the data obtained, the glucose catabolism in H. saccharophilum, A. fermentum, and A. tropicus mainly proceeds via the fructose bisphosphate pathway. The pH optima of the key enzymes of the glucose metabolism of the alkaliphiles are shifted to alkaline values. In A. tropicus, formate is formed from pyruvate under the action of pyruvate formate-lyase; and in the haloanaerobe H. saccharophilum, formate dehydrogenase is involved in formate metabolism.     

Identification and purification of a distinct dihydrolipoamide dehydrogenase from pea chloroplasts

Two distinct dihydrolipoamide dehydrogenases (E3s, EC 1.8.1.4) have been detected in pea (Pisum sativum L. cv. Little Marvel) leaf extracts and purified to at or near homogeneity. The major enzyme, a homodimer with an apparent subunit M_r value 56 000 (80–90% of overall activity), corresponded to the mitochondrial isoform studied previously, as confirmed by electrospray mass spectrometry and N-terminal sequence analysis. The minor activity (10–20%), which also behaved as a homodimer, copurified with chloroplasts, and displayed a lower subunit M_r value of 52 000 which was close to the M_r value of 52 614±9.89 Da determined by electrospray mass spectrometry. The plastidic enzyme was also present at low levels in root extracts where it represented only 1–2% of total E3 activity. The specific activity of the chloroplast enzyme was three-to fourfold lower than its mitochondrial counterpart. In addition, it displayed a markedly higher affinity for NAD⁺ and was more sensitive to product inhibition by NADH. It exhibited no activity with NADP⁺ as cofactor nor was it inhibited by the presence of high concentrations of NADP⁺ or NADPH. Antibodies to the mitochondrial enzyme displayed little or no cross-reactivity with its plastidic counterpart and available amino acid sequence data were also suggestive of only limited sequence similarity between the two enzymes. In view of the dual location of the pyruvate dehydrogenase multienzyme complex (PDC) in plant mitochondria and chloroplasts, it is likely that the distinct chloroplastic E3 is an integral component of plastidic PDC, thus representing the first component of this complex to be isolated and characterised to date.Abbreviations E1 pyruvate dehydrogenase - E2 dihydrolipoamide acetyltransferase - E3 dihydrolipoamide dehydrogenase - PDC pyruvate dehydrogenase complex - OGDC 2-oxoglutarate dehydrogenase complex - GDC glycine decarboxylase complex - SDS-PAGE sodium dodecyl sulphate/polyacrylamide gel electrophoresis - TDP thiamine diphosphate - M_r relative molecular mass J.G.L. is grateful to the Biotechnology and Biological Sciences Research Council (BBSRC), U.K. for continuing financial support. M.C. is the holder of a BBSRC-funded earmarked Ph.D. studentship.     

Localization of the enzymes involved in H2 and formate metabolism inSyntrophospora bryantii

Cell-free extracts of crotonate-grown cells of the syntrophic butyrate-oxidizing bacteriumSyntrophospora bryantii contained high hydrogenase activities (8.5–75.8 µmol · min^–1 mg^–1 protein) and relatively low formate dehydrogenase activities (0.04–0.07 µmol · min^–1 mg^–1 protein). The K _M value and threshold value of the hydrogenase for H₂ were 0.21 mM and 18 µM, respectively, whereas the K _M value and threshold value of the formate dehydrogenase for formate were 0.22 mM and 10 µM, respectively. Hydrogenase, butyryl-CoA dehydrogenase and 3-OH-butyryl-CoA dehydrogenase were detected in the cytoplasmic fraction. Formate dehydrogenase and CO₂ reductase were membrane-bound, likely located at the outer aspect of the cytoplasmic membrane. Results suggest that during syntrophic butyrate oxidation H₂ is formed intracellularly while formate is formed at the outside of the cell.     

Pyruvate fermentation in light-grown cells ofRhodospirillum rubrum during adaptation to anaerobic dark conditions

Pyruvate fermentation inRhodospirillum rubrum (strains F₁, S₁, and Ha) was investigated using cells precultured on different substrates anaerobically in the light and than transferred to anaerobic dark conditions. Pyruvate formate lyase was always the key enzyme in pyruvate fermentation but its activity was lower than in cells which have been precultured aerobically in darkness. The preculture substrate also had a clear influence on the pyruvate formate lyase activity. Strains F₁ and S₁ metabolized the produced formate further to H₂ and CO₂. A slight production of CO₂ from pyruvate, without additional H₂-production, could also be detected. It was concluded from this that under anaerobic dark conditions a pyruvate dehydrogenase was also functioning. On inhibition of pyruvate formate lyase the main part of pyruvate breakdown was taken over by pyruvate dehydrogenase.When enzyme synthesis was inhibited by chloramphenicol, propionate production in contrast to formate production was not affected. Protein synthesis was not significant during anaerobic dark culture. Bacteriochlorophyll. however, showed, after a lag phase, a clear rise.Abbreviations Bchl Bacteriochlorophyll - CoA Coenzyme A - DSM Deutsche Sammlung von Mikroorganismen (Göttingen) - OD optical density - PHBA poly--hydroxybutyric acid - R Rhodospirillum     

Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L.

The maximum extractable activities of twenty-one photosynthetic and glycolytic enzymes were measured in mature leaves of Mesembryanthemum crystallinum plants, grown under a 12 h light 12 h dark photoperiod, exhibiting photosynthetic characteristics of either a C₃ or a Crassulacean acid metabolism (CAM) plant. Following the change from C₃ photosynthesis to CAM in response to an increase in the salinity of in the rooting medium from 100 mM to 400 mM NaCl, the activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) increased about 45-fold and the activities of NADP malic enzyme (EC 1.1.1.40) and NAD malic enzyme (EC 1.1.1.38) increased about 4- to 10-fold. Pyruvate, Pi dikinase (EC 2.7.9.1) was not detected in the non-CAM tissue but was present in the CAM tissue; PEP carboxykinase (EC 4.1.1.32) was detected in neither tissue. The induction of CAM was also accompanied by large increases in the activities of the glycolytic enzymes enolase (EC 4.2.1.11), phosphoglyceromutase (EC 2.7.5.3), phosphoglycerate kinase (EC 2.7.2.3), NAD glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and glucosephosphate isomerase (EC 2.6.1.2). There were 1.5- to 2-fold increases in the activities of NAD malate dehydrogenase (EC 1.1.1.37), alanine and aspartate aminotransferases (EC 2.6.1.2 and 2.6.1.1 respectively) and NADP glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). The activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphatase (EC 3.1.3.11), phosphofructokinase (EC 2.7.1.11), hexokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) remained relatively constant. NADP malate dehydrogenase (EC 1.1.1.82) activity exhibited two pH optima in the non-CAM tissue, one at pH 6.0 and a second at pH 8.0. The activity at pH 8.0 increased as CAM was induced. With the exceptions of hexokinase and glucose-6-phosphate dehydrogenase, the activities of all enzymes examined in extracts from M. crystallinum exhibiting CAM were equal to, or greater than, those required to sustain the maximum rates of carbon flow during acidification and deacidification observed in vivo. There was no day-night variation in the maximum extractable activities of phosphoenolpyruvate carboxylase, NADP malic enzyme, NAD malic enzyme, fructose-1,6-bisphosphatase and NADP malate dehydrogenase in leaves of M. crystallinum undergoing CAM.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

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.