C nuclear magnetic resonance studies of propionate catabolism in methanogenic cocultures

Propionate catabolism was monitored in anaerobic cocultures of propionate-degrading and methanogenic bacteria. Metabolism was monitored by use of C-enriched propionate and succinate. The intermediates identified indicated that the methylmalonyl coenzyme A pathway was used in these cultures. The data also indicated that a transcarboxylation reaction between succinate and propionyl coenzyme A occurred, yielding propionate and methylmalonyl coenzyme A.     

The presence and possible function of methylmalonyl CoA mutase and propionyl CoA carboxylase in Spirometra mansonoides.

Both spargana and adult forms of Spirometra mansonoides were shown to accumulate lactate, succinate, acetate, and propionate upon in vitro incubation. Adults differed markedly from the spargana in that quantitatively the most significant products of the former were acetate and propionate while the latter formed primarily acetate and lactate. The adults accumulated approximately 32 times more propionate than the spargana per gram of tissue. In accord with this propionate formation, propionyl CoA carboxylase and methylmalonyl CoA mutase have been found to be present in both stages of the parasite. As might be predicted, however, the activities of the carboxylase and mutase were 100-fold and 10-fold higher, respectively, in the adults as compared to the larvae. A possible physiological relationship between propionate formation and energy generation is suggested. Accordingly, inorganic 32P was incorporated into ATP upon incubation of methylmalonyl CoA with a homogenate obtained from adult S. mansonoides. Since methylmalonyl CoA mutase requires vitamin B12 coenzyme, a relationship between vitamin B12 content and propionate formation in helminths is suggested.     

Regulation of carbon and electron flow in Propionispira arboris: relationship of catabolic enzyme levels to carbon substrates fermented during propionate formation via the methylmalonyl coenzyme A pathway.

A detailed study of the glucose fermentation pathway and the modulation of catabolic oxidoreductase activities by energy sources (i.e., glucose versus lactate or fumarate) in Propionispira arboris was performed. 14C radiotracer data show the CO2 produced from pyruvate oxidation comes exclusively from the C-3 and C-4 positions of glucose. Significant specific activities of glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-bisphosphate aldolase were detected, which substantiates the utilization of the Embden-Meyerhoff-Parnas path for glucose metabolism. The methylmalonyl coenzyme A pathway for pyruvate reduction to propionate was established by detection of significant activities (greater than 16 nmol/min per mg of protein) of methylmalonyl coenzyme A transcarboxylase, malate dehydrogenase, and fumarate reductase in cell-free extracts and by 13C nuclear magnetic resonance spectroscopic demonstration of randomization of label from [2-13C]pyruvate into positions 2 and 3 of propionate. The specific activity of pyruvate-ferredoxin oxidoreductase, malate dehydrogenase, fumarate reductase, and transcarboxylase varied significantly in cells grown on different energy sources. D-Lactate dehydrogenase (non-NADH linked) was present in cells of P. arboris grown on lactate but not in cells grown on glucose or fumarate. These results indicate that growth substrates regulate synthesis of enzymes specific for the methylmalonyl coenzyme A path and initial substrate transformation.     

Dissimilation of Methionine by Achromobacter starkeyi

Methionine was decomposed by some bacteria which were isolated from soil. The sulfur of the methionine was liberated as methanethiol, and part of this became oxidized to dimethyl disulfide. Detailed studies with one of these cultures, Achromobacter starkeyi, indicated that the first step in methionine decomposition was its oxidadative deamination to α-keto-γ-methyl mercaptobutyrate by a constitutive amino acid oxidase. The following steps were carried out by inducible enzymes, the synthesis of which was inhibited by chloramphenicol. α-Keto-γ-methyl mercaptobutyrate was split producing methanethiol and α-keto butyrate which was oxidized to propionate. The metabolism of propionate was similar to that described for animal tissues; the propionate was carboxylated to succinate via methyl malonyl coenzyme A, and the succinate was metabolized through the Krebs cycle.     

Discovering new enzymes and metabolic pathways: conversion of succinate to propionate by Escherichia coli

The Escherichia coli genome encodes seven paralogues of the crotonase (enoyl CoA hydratase) superfamily. Four of these have unknown or uncertain functions; their existence was unknown prior to the completion of the E. coli genome sequencing project. The gene encoding one of these, YgfG, is located in a four-gene operon that encodes homologues of methylmalonyl CoA mutases (Sbm) and acyl CoA transferases (YgfH) as well as a putative protein kinase (YgfD/ArgK). We have determined that YgfG is methylmalonyl CoA decarboxylase, YgfH is propionyl CoA:succinate CoA transferase, and Sbm is methylmalonyl CoA mutase. These reactions are sufficient to form a metabolic cycle by which E. coli can catalyze the decarboxylation of succinate to propionate, although the metabolic context of this cycle is unknown. The identification of YgfG as methylmalonyl CoA decarboxylase expands the range of reactions catalyzed by members of the crotonase superfamily.     

Biosynthesis of methyl branched hydrocarbons of the German cockroach Blattella germanica (L.) (orthoptera,blattellidae)

The precursors and directionality of synthesis of the methyl branched cuticular hydrocarbons and the female contact sex pheromone, 3,11-dimethyl-2-nonacosanone, of the German cockroach, Blattella germanica, were investigated by radiotracer and carbon-13 NMR techniques. The amino acids [G-³H]valine, [4,5-³H]isoleucine and [3,4-¹⁴C₂]methionine labeled the hydrocarbon fraction in a manner indicating that the carbon skeletons of all three amino acids serve as the methyl branch group donor. The incorporation of [1,4-¹⁴C₂]- and [2,3-¹⁴C₂]succinates into the hydrocarbon and acylglycerol/polar lipid fractions indicated that succinate also served as a precursor to methylmalonyl-CoA. Carbon-13 NMR analyses showed that [1-¹³C]propionate labeled the carbon adjacent to the tertiary carbon, and, for the 3,x-dimethylalkanes, that carbon-4 and not carbon-2 was enriched. [1-¹³C]Acetate labeled carbon-2 of these hydrocarbons. This indicates that the methyl branching groups of the 3,x-dimethylalkanes were inserted early in the chain elongation process. [3,4,5-¹³C₃]Valine labeled the methyl, tertiary and carbon adjacent to the tertiary carbon of the methyl branched alkanes. Thus, the methyl branched hydrocarbon was formed by the insertion of methylmalonyl units derived from propionate, isoleucine, valine, methionine and succinate early in chain elongation.     

Succinate metabolism related to lipid synthesis in the housefly Musca domestica L

The metabolism of succinate was examined in the housefly Musca domestica L. The labeled carbons from [2,3-¹⁴C]succinate were readily incorporated into cuticular hydrocarbon and internal lipid, whereas radioactivity from [1,4-¹⁴C]succinate was not incorporated into either fraction. Examination of the incorporation of [2,3-¹⁴C]succinate, [1-¹⁴C]acetate, and [U-¹⁴C]proline into hydrocarbon by radio-gas-liquid chromatography showed that each substrate gave a similar labeling pattern, which suggested that succinate and proline were converted to acetyl-CoA prior to incorporation into hydrocarbons. Carbon-13 nuclear magnetic resonance showed that the labeled carbons from [2,3-¹³C]succinate enriched carbons 1, 2, and 3 of hydrocarbons with carbon-carbon coupling showing that carbons 2 and 3 of succinate were incorporated as an intact unit. Radio-high-performance liquid chromatographic analysis of [2,3-¹⁴C]succinate metabolism by mitochondrial preparations showed that in addition to labeling fumarate, malate, and citrate, considerable radioactivity was also present in the acetate fraction. The data show that succinate was not converted to methylmalonate and did not label hydrocarbon via a methylmalonyl derivative. Malic enzyme was assayed in sonicated mitochondria prepared from the abdomens and thoraces of 1- and 4-day-old insects; higher activity was obtained with NAD⁺ in mitochondria prepared from thoraces, whereas NADP⁺ gave higher activity with abdomen preparations. These data document the metabolism of succinate to acetyl-CoA and not to a methylmalonyl unit prior to incorporation into lipid in the housefly and establish the role of the malic enzyme in this process.     

Gut tract microorganisms supply the precursors for methyl-branched hydrocarbon biosynthesis in the termite,Zootermopsis nevadensis

In vivo and in vitro experiments were performed to examine the role of succinate and other potential precursors of the methylmalonyl-CoA used for methyl-branched hydrocarbon biosynthesis in the termite Zootermopsis nevadensis. The in vivo incorporation of [1,4-¹⁴C]succinate and [2,3-¹⁴C]succinate into hydrocarbon confirmed that succinate is a direct precursor to the methyl branch unit. The other likely precursors, the branched chain amino acids valine and isoleucine, were not efficiently incorporated into hydrocarbon. Carbon-13 NMR showed that one of the labeled carbons of [1,4-¹³C]succinate labeled position 6 of 5-methylalkanes and positions 6 and 18 of 5,17-dimethylalkanes, indicating that succinate, as a methylmalonyl-CoA unit, was incorporated as the third unit to form 5-methylheneicosane and as both the third and ninth units to form 5,17-dimethylheneicosane. Analysis of organic acids after the in vivo metabolism of [2,3-¹⁴C]succinate showed that succinate was converted to propionate and methylmalonate. Labeled succinate injected into the hemolymph was readily taken up by the gut tract. Isolated gut tissue efficiently converted succinate to acetate and propionate, both of which were released into the incubation media. Mitochondria from termite tissue (minus gut tract) converted succinate to methylmalonate and propionate only in the presence of malonic acid, an inhibitor of succinate dehydrogenase. The results of these studies show that while termite mitochondria are able to convert succinate to propionate and methylmalonate, most of the propionate used for methyl-branched hydrocarbon biosynthesis is produced by gut tract microorganisms. The propionate is then presumably transported through the hemolymph to epidermal cells for use in methyl-branched hydrocarbon biosynthesis.     

Metabolism of Glutamate in Purple Nonsulfur Bacteria Participation of Vitamin B12

Purple nonsulfur bacteria, Rhodospirillum rubrum and Rhodopseudomonas spheroides were found to possess coenzyme B₁₂-dependent glutamate mutase activity. Cell-free extracts of these bacteria grown on Co²⁺-containing media catalyzed the conversion of glutamate to β-methylaspartate and further to mesaconate. The activity of the cell-free extracts of these organisms cultivated on Co²⁺-deficient media was markedly lower than that of the normal cells. Addition of coenzyme B₁₂ to the former reaction mixture enhanced the mesaconate formation via β-methylaspartate. These results indicate the involvement of coenzyme Independent glutamate mutase of these bacteria in the dissimilation of glutamate to acetyl-CoA and pyruvate through the following pathway.

glutamate→β→methylaspartate→mesaconate→citramalate→→acetyl-CoA, pyruvate On the other hand, a greater part of glutamate was converted to α-hydroxyglutarate and succinate with the cell-free extracts of these photosynthetic bacteria. This fact, taking account of the presence of propionyl-CoA carboxylase in these bacteria, implies the participation of coenzyme B₁₂-dependent (R)-methylmalonyl-CoA mutase in the formation of succinate via the following route.

glutamate→α-ketoglutarate→α-hydroxyglutarate→propionate→propionyl-CoA→(S)-methylmalonyl-CoA→(R)-methylmalonyl-CoA→succinyl-CoA     

Biogenesis of flavoprotein and cytochrome components in hepatic mitochondria from riboflavin-deficient rats

Using difference spectrophotometry, measurements of succinate dehydrogenase activity, and SDS-polyacrylamide gels, the biochemical properties of hepatic mitochondria from riboflavin-deficient rats were monitored during recovery on riboflavin. [¹⁴C]Riboflavin was incorporated into four mitochondrial flavoproteins having covalently bound flavin coenzyme. Alterations in cytochromes, especially cytochrome oxidase, and the biosyntheses of succinate dehydrogenase, monoamine oxidase, sarcosine dehydrogenase, and an unknown flavoprotein were observed.     

Metabolic Pathway for Propionate Utilization by Phosphorus-Accumulating Organisms in Activated Sludge: 13C Labeling and In Vivo Nuclear Magnetic Resonance

In vivo ¹³C and ³¹P nuclear magnetic resonance techniques were used to study propionate metabolism by activated sludge in enhanced biological phosphorus removal systems. The fate of label supplied in [3-¹³C]propionate was monitored in living cells subjected to anaerobic/aerobic cycles. During the anaerobic phase, propionate was converted to polyhydroxyalkanoates (PHA) with the following monomer composition: hydroxyvalerate, 74.2%; hydroxymethylvalerate, 16.9%; hydroxymethylbutyrate, 8.6%; and hydroxybutyrate, 0.3%. The isotopic enrichment in the different carbon atoms of hydroxyvalerate (HV) produced during the first anaerobic stage was determined: HV₅, 59%; HV₄, 5.0%; HV₃, 1.1%; HV₂, 3.5%; and HV₁, 2.8%. A large proportion of the supplied label ended up on carbon C-5 of HV, directly derived from the pool of propionyl-coenzyme A (CoA), which is primarily labeled on C-3; useful information on the nature of operating metabolic pathways was provided by the extent of labeling on C-1, C-2, and C-4. The labeling pattern on C-1 and C-2 was explained by the conversion of propionyl-CoA to acetyl-CoA via succinyl-CoA and the left branch of the tricarboxylic acid cycle, which involves scrambling of label between the inner carbons of succinate. This constitutes solid evidence for the operation of succinate dehydrogenase under anaerobic conditions. The labeling in HV₄ is explained by backflux from succinate to propionyl-CoA. The involvement of glycogen in the metabolism of propionate was also demonstrated; moreover, it was shown that the acetyl moiety to the synthesis of PHA was derived preferentially from glycogen. According to the proposed metabolic scheme, the decarboxylation of pyruvate is coupled to the production of hydrogen, and the missing reducing equivalents should be derived from a source other than glycogen metabolism.     

Autotrophic CO2 fixation in Chlorobium limicola. Evidence for the operation of a reductive tricarboxylic acid cycle in growing cells

Chlorobium limicola was grown on a mineral salts medium with CO₂ as the main carbon source supplemented with specifically labeled ¹⁴C propionate and the incorporation of ¹⁴C into alanine ( intracellular pyruvate), aspartate ( oxaloacetate), and glutamate ( -ketoglutarate) was studied in long term labeling experiments. During growth in presence of propionate 30% of the cell carbon were derived from propionate and 70% from CO₂. Propionate was not oxidized to CO₂.All three amino acids were found to be labeled. The labeling patterns indicate that propionate was assimilated via propionyl CoA, methylmalonyl CoA and succinyl CoA. When 1-¹⁴C propionate was the labeled precursor no radioactivity was found in the carboxyl group(s) of alanine, aspartate and glutamate, excluding the incorporation of propionate into the amino acids via succinate oxidation to fumarate. With 1-¹⁴C propionate preferentially aspartate (C-3) and glutamate (C-2) became labeled, with 2-¹⁴C propionate alanine (C-3) and glutamate (C-4). These findings indicate that propionate was incorporated into the amino acids via succinyl CoA, -ketoglutarate, isocitrate, and citrate, followed by a si-type cleavage of citrate to oxaloacetate and acetyl CoA (or acetate). Similar experiments with U-¹⁴C acetate confirm these conclusions. Thus, all reactions of the proposed reductive tricarboxylic acid cycle could be demonstrated in autotrophically growing cells.     

The anaerobic formation of propionic acid in the mitochondria of the lugwormArenicola marina

Summary The anaerobic transformation of malate and succinate into propionate was demonstrated in homogenates and mitochondria isolated from the body wall musculature ofArenicola marina, a facultative anaerobic polychaete. Synthesis of propionate from succinate was enhanced by the addition of malate and ADP. In the presence of malate, acetate was formed in addition to propionate. Maximal quantities of both fatty acids were produced by mitochondria incubated with malate, succinate, and ADP. Since the rate of propionate production in this case was about the same as in homogenates when related to fresh weight, it is concluded that the enzymatic system involved is localized exclusively in the mitochondria. The rate of propionate production is correlated with the concentration of succinate, saturation being reached at about 5 mM. In tracer experiments using (methyl-¹⁴C)-malonyl-CoA, 2,3-¹⁴C-succinate, and 1-¹⁴C-propionate as precursors, the pathway of the transformation of succinate into propionate was examined. The results indicate that methylmalonyl-CoA is an intermediary product. It was shown that the synthesis of propionate from succinate is coupled to the formation of ATP. The ratio ATP/propionate was 0.76. Dinitrophenol had only a slight effect on this ratio, although the utilization of succinate was inhibited considerably. It is concluded that in vivo substrate level phosphorylation occurs equimolar to the formation of propionate from succinate.Abbreviations Ap ₅ A P₁,P₅-di(adenosine-5-)pentaphosphate - DNP 2,4-dinitrophenol - mma methylmalonic acid - mm-CoA methylmalonyl-CoA Enzymes EC 6.2.1.1 Acetate thiokinase (AMP) - EC 3.6.1.3 actomyosin ATPase - EC 2.7.4.3 adenylate kinase - EC 2.8.3.1 CoA transferase - EC 2.7.1.1 hexokinase - EC 2.1.3.1 methylmalonyl-CoA carboxyltransferase - EC 5.4.99.1 methylmalonyl-CoA isomerase - EC 5.1.99.1 methylmalonyl-CoA racemase - EC 6.4.1.3 propionyl-CoA carboxylase - EC 1.2.4.1 pyruvate dehydrogenase Supported by Deutsche Forschungsgemeinschaft Gr 456/6     

Pathway of propionate formation in Desulfobulbus propionicus

Whole cells of Desulfobulbus propionicus fermented [1-¹³C]ethanol to [2-¹³C] and [3-¹³C]propionate and [1-¹³C]-acetate, which indicates the involvement of a randomizing pathway in the formation of propionate. Cell-free extracts prepared from cells grown on lactate (without sulfate) contained high activities of methylmalonyl-CoA: pyruvate transacetylase, acetase kinase and reasonably high activities of NAD(P)-independent L(+)-lactate dehydrogenase NAD(P)-independent pyruvate dehydrogenase, phosphotransacetylase, acetate kinase and reasonably high activity of NAD(P)-independent L(+)-lactate dehydrogenase, fumarate reductase and succinate dehydrogenase. Cell-free extracts catalyzed the conversion of succinate to propionate in the presence of pyruvate, CoA and ATP and the oxaloacetate-dependent conversion of propionate to succinate. After growth on lactate or propionate in the presence of sulfate similar enzyme levels were found except for fumarate reductase which was considerably lower. Fermentative growth on lactate led to higher cytochrome b contents than growth with sulfate as electron acceptor.The labeling studies and the enzyme measurements demonstrate that in Desulfobulbus propionate is formed via a succinate pathway involving a transcarboxylase like in Propionibacterium. The same pathway may be used for the degradation of propionate to acetate in the presence of sulfate.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PEP phosphoenolpyruvate     

Separation and functions of two acyl CoA transferases from Ascaris lumbricoides mitochondria

Many invertebrates accumulate propionate, or products derived from propionate, as products of fermentation. Evidence has been reported that the nematode, Ascaris suum, the cestode, Spirometra mansonoides, and the trematode, Fasciola hepatica, accumulate propionate by means of an adenosine triphosphate (ATP)-generating decarboxylation of succinate. To generate energy, an acyl coenzyme A (CoA) transferase that would transfer CoA to succinate is required as one component of the sequence of reactions. Recently, an acyl CoA transferase was isolated from Ascaris mitochondria and purified to electrophoretic homogeneity. However, upon examination of the substrate specificities of this enzyme, it was found essentially to lack the ability to use succinate or succinyl CoA as an acceptor or donor of CoA, respectively. Therefore, this transferase could not serve to activate succinate. This article describes the isolation of an additional acyl CoA transferase from Ascaris mitochondria that appears to be unique in its substrate specificity and that could easily account not only for the activation of succinate but also for the regulation of succinate metabolism primarily in the direction of decarboxylation to propionate. This is in contrast with mammalian tissues, which act in the opposite direction by catalyzing the fixation of CO2 into propionate, thereby forming succinate and accounting for the glycogenic nature of dietary propionate. Possible functions of the two acyl CoA transferases are discussed.     

Congenital methylmalonic acidemia: a variant of the B12 'non-responsive' form with evidence for reduced affinity of methylmalonyl-CoA mutase for its B12-coenzyme.

Methylmalonate metabolism was investigated in fibroblasts and leukocytes of two unrelated patient with a B12-nonresponsive type of congenital methylmalonic acidemia. Intact fibroblasts from both patients showed a defective metabolism of methyl-14 c-malonate to 14CO2, whereas no such defect was found in their intact peripheral leukocytes. In disrupted fibroblasts, the conversion of methylmalonyl coenzyme A to succinyl coenzyme A was markedly reduced but was completely normalized by the addition of 5'-deoxyadenosylcobalamin (AdoCb1; 10(-5) mol/l), the specific coenzyme of methylmalonyl coenzyme A mutase. Assays with decreasing concentrations of AdoCbl (10(-5)-10(-11) mol/l) suggested a reduced affinity of the mutase apoenzyme for its coenzyme, implicating yet another variant of this heterogeneous disease.     

Metabolic pathway to propionate of Pectinatus frisingensis,a strictly anaerobic beer-spoilage bacterium

      Pectinatus frisingensis, a recently described species of anaerobic mesophilic beer-spoilage bacteria, grows by fermenting various organic compounds, and produces mainly propionate, acetate, and succinate. Although acrylate and succinate were both dismutated by dense resting-cell suspensions, propionate production proceeded through the succinate pathway: [3-¹³C]pyruvate consumption led to equal ¹³C-labeling of propionate on methyl and methylene groups. Growth on glucose or glycerol led to a similar propionate to acetate ratio, suggesting dihydroxyacetone phosphate as being a common metabolic intermediate. Diacetyl, 1,3-propanediol, and 2,3-butanediol were not growth substrates or fermentation products, but they were all dismutated by dense resting-cell suspensions to acetate and propionate. Acetoin was a minor fermentation product. The consumption of [2-¹³C] or [3-¹³C]pyruvate by dense resting-cell suspensions demonstrated the involvement of two equivalent pyruvate molecules during acetoin production. Key enzymes involved in this metabolism were measured in anoxic cell-free extracts. A tentative metabolic pathway to the main fermentation products was proposed from the above results. Received: 17 February 1994 / Accepted: 30 August 1994     

Rapid prenatal and postnatal detection of inborn errors of propionate,methylmalonate, and cobalamin metabolism: A sensitive assay using cultured cells

Summary A sensitive, reliable, and easily performed procedure is described for the prenatal and postnatal detection of inborn errors of propionate, methylmalonate, and cobalamin metabolism using cultured amniotic cells and skin fibroblasts. With this assay, control fibroblast lines incorporated a mean of 6.89 nanoatoms ¹⁴C/mg protein from [1-¹⁴C]propionate into trichloroacetic acid (TCA)-precipitable cell material in 10h. Twenty-five mutant fibroblast lines from patients with propionicacidemia or one of the methylmalonicacidemias fixed 0.04 to 0.93 nanoatoms ¹⁴C/mg. Considerable variation was observed, both among and within discrete mutant classes, with respect to the residual amount of propionate pathway activity, possibly reflecting further molecular heterogeneity in these disorders.We applied this procedure to cultured amniotic cells from controls and 4 midtrimester pregnancies at risk for methylmalonicacidemia and diagnosed one fetus with a methylmalonyl CoA apomutase defect and 3 fetuses which were unaffected.Presented in part at the annual meeting of the Society for Pediatric Research, St. Louis, Missouri, April 1976.