13C nuclear magnetic resonance studies on bacteriochlorophyll a biosynthesis in Rhodopseudomonas spheroides S

The 13C NMR spectra were analyzed in bacteriochlorophyll a and magnesium protoporphyrin methyl ester formed in Rhodopseudomonas spheroides S. in the presence of L-[1-13C]glutamate and [2-13C]glycine. After reassignment of three alpha-pyrrolic carbons (C-9, -14 and -16) of bacteriochlorophyll a, the spectra showed that C-2 of glycine was preferentially incorporated into the eight-carbon atoms in these tetrapyrrole macrocycles derived from C-5 of 5-aminolevulinic acid (ALA). C-2 of glycine was also incorporated specifically into methyl ester carbon of magnesium protoporphyrin IX methyl ester and methoxyl carbon of methoxycarbonyl group attached to isocyclic ring of bacteriochlorophyll a. No enrichment of these nine-carbon atoms was observed in the spectrum of bacteriochlorophyll formed in the presence of L-[1-13C]glutamate, showing exclusive operation of ALA synthase on bacteriochlorophyll biosynthesis.     

13C-NMR evidence of bacteriochlorophyll a formation by the C5 pathway in Chromatium

The 13C-NMR spectra of bacteriochlorophyll a formed in the presence of L-[1-13C]glutamate and [2-13C]glycine in Chromatium vinosum strain D were analyzed. The isotope in the glutamate was specifically incorporated into eight carbon atoms in the tetrapyrrole macrocycle derived from the C-5 of 5-aminolevulinic acid (ALA), and the 13C in glycine was incorporated into the methyl carbon of the methoxycarbonyl group attached to the isocyclic ring of bacteriochlorophyll a. These labeling patterns provide evidence for the exclusive operation of the C5 pathway in ALA biosynthesis in the bacterium. The 13C chemical shifts of two quaternary carbons (C-9 and C-16) of bacteriochlorophyll a were reassigned in the present study.     

Two Biosynthetic Pathways to delta-Aminolevulinic Acid in a Pigment Mutant of the Green Alga, Scenedesmus obliquus

Pigment mutant C-2A′ of the unicellular green alga Scenedesmus obliquus develops only traces of chlorophyll and has no detectable amount of δ-aminolevulinic acid (ALA) when grown in the dark. In light it develops ALA and in the presence of levulinic acid (LA), a competitive inhibitor of ALA dehydratase, it accumulates 0.18 mmoles of ALA per 10 microliters of packed cell volume per 12 hours. This amount could be increased up to 15 times by feeding precursors and cofactors.

Incubation with [U-¹⁴C]glutamate, [1-¹⁴C]glutamate, and [2-¹⁴C]glycine yielded significantly labeled ALA, whereas [1-¹⁴C]glycine did not label the ALA specifically. Thus, two pathways using either glycine/succinyl-coenzyme A or incorporating the whole C-5-skeleton of glutamate into ALA are present in this alga. The efficiency of the glycine/succinyl-coenzyme A pathway seems to be three times higher than that of the glutamate pathway. Incubation with [5-¹⁴C]2-ketoglutarate, which can serve both pathways as a precursor, resulted in radioactivity of ALA as high as the sum of both labeling with [1-¹⁴C]glutamate and [2-¹⁴C]glycine.

Since the newly synthesized chlorophyll was radioactive regardless of labeled substrate employed, both pathways culminate in chlorophyll formation.

     

On the biosynthesis of 5-hydroxybenzimidazolylcobamide (vitamin B12-factor III) in Methanosarcina barkeri

Exogenous 5-hydroxy-[2-¹⁴C]benzimidazole was transformed by Methanosarcina barkeri into 5-hydroxy-[2-¹⁴C]benzimidazolylcobamide. Thereby the endogenous biosynthesis of 5-hydroxybenzimidazole was completely blocked.Benzimidazole and 5,6-dimethylbenzimidazole were used by M. barkeri to form benzimidazolylcobamide respectively 5,6-dimethylbenzimidazolylcobamide (vitamin B₁₂), but in these cases the endogenous biosynthesis of factor III was not completely suppressed.With [2-¹⁴C]benzimidazole it was demonstrated that this base as well as the benzimidazolylcobamide formed thereof are no precursors in the biosynthesis of 5-hydroxybenzimidazolylcobamide.Glycine instead was found to be a building block for the biosynthesis of 5-hydroxybenzimidazole, since radioactivity from [1-¹⁴C] and [2-¹⁴C]glycine was incorporated, into the base moiety of factor III, but not into its corrin moiety. With [1-¹³C]glycine and ¹³C-NMR-spectroscopy it was shown that C-1 of glycine gets C-3a of 5-hydroxybenzimidazole.[1-¹³C]glycine also led to a single prominent signal in the ¹³C-NMR-spectrum of coenzyme F₄₂₀, this was assigned to C-10a.Thus C-1 of glycine was incorporated into the hydroxybenzene part of 5-hydroxybenzimidazole, whereas it was not incorporated into this part of coenzyme F₄₂₀, indicating that the hydroxybenzene part of these two compounds is not formed from a common intermediate. L-[U-¹⁴C]glutamate led to the exclusive labeling of the corrin ring of factor III, showing that the corrin precursor 5-aminolevulinic acid is formed by the C-5 pathway in M. barkeri.These experiments indicate that the biosynthesis of factor III in the archaebacterium M. barkeri is similar to the corrinoid biosynthesis in the anaerobic eubacteria Eubacterium limosum, Clostridium barkeri, and Clostridium thermoaceticum.     

Biosynthesis of artemisinin – revisited

Artemisinin is a well-known antimalarial drug isolated from the Artemisia annua plant. The biosynthesis of this well-known molecule has been reinvestigated by using [1-¹³C]acetate, [2-¹³C]acetate, and [1,6-¹³C₂]glucose. The ¹³C peak enrichment in artemisinin was observed in six and nine carbon atoms from [1-¹³C]acetate and [2-¹³C]acetate, respectively. The ¹³C NMR spectra of ¹³C-enriched artemisinin suggested that the mevalonic acid (MVA) pathway is the predominant route to biosynthesis of this sesquiterpene. On the other hand, the peak enrichment of five carbons of ¹³C-artemisinin including carbon atoms originating from methyls of dimethylallyl group of geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP) was observed from [1,6-¹³C₂]glucose. This suggested that GPP which is supposed to be biosynthesized in plastids travels from plastids to cytosol through the plastidial wall and combines with isopentenyl pyrophosphate (IPP) to form the (E,E)-FPP which finally cyclizes and oxidizes to artemisinin. In this way the DXP pathway also contributes to the biosynthesis of this sesquiterpene.     

The fermentation of xylose: studies by carbon-13 nuclear magnetic resonance spectroscopy

Summary The fermentation ofd-xylose byPachysolen tannophilus, Candida shehatae, andPichia stipitis has been investigated by¹³C-nuclear magnetic resonance spectroscopy of both whole cells and extracts. The spectra of whole cells metabolizingd-xylose with natural isotopic abundance had significant resonance signals corresponding only to xylitol, ethanol and xylose. The spectra of whole cells in the presence of [1-¹³C]xylose or [2-¹³C]xylose had resonance signals corresponding to the C-1 or C-2, respectively, of xylose, the C-1 or C-2, respectively, of xylitol, and the C-2 or C-1, respectively, of ethanol. Xylitol was metabolized only in the presence of an electron acceptor (acetone) and the only identifiable product was ethanol. The fact that the amount of ethanol was insufficient to account for the xylitol metabolized indicates that an additional fate of xylitol carbon must exist, probably carbon dioxide. The rapid metabolism of xylulose to ethanol, xylitol and arabinitol indicates that xylulose is a true intermediate and that xylitol dehydrogenase catalyzes the reduction (or oxidation) with different stereochemical specificity from that which interconverts xylitol andd-xylulose. The amino acidl-alanine was identified by the resonance position of the C-3 carbon and by enzymatic analysis of incubation mixtures containing yeast and [1-¹³C]xylose or [1-¹³C]glucose. The position of the label from both substrates and the identification of isotope also in C-1 of alamine indicates flux through the transketolase/transaldolase pathway in the metabolism. The identification of a resonance signal corresponding to the C-1 of ethanol in spectra of yeast in the presence of [1-¹³C]xylose and fluoroacetate (but not arsenite) indicates the existence of equilibration of some precursor of ethanol (e.g. pyruvate) with a symmetric intermediate (e.g. fumarate or succinate) under these conditions.     

Biosynthesis of Bisorbicillinoid in Trichoderma sp. USF-2690; Evidence for the Biosynthetic Pathway,via Sorbicillinol,of Sorbicillin,Bisorbicillinol, Bisorbibutenolide,and…

An incorporation study of [1-¹³C] and [1,2-¹³C₂] labeled sodium acetates into sorbicillinol 1 established a ring closure system between C-1 and C-6 and the positions that were oxidized and/or methylated on a hexaketide chain. Subsequent investigations, using ¹³C-labeled 1 prepared from [1-¹³C] labeled sodium acetate, clearly demonstrated that both bisorbicillinol 2 and sorbicillin 6 incorporated ¹³C-labeled 1 into their carbon skeletons. ¹³C-labeled bisorbicillinols 2 derived from [1-¹³C]- and [2-¹³C]-labeled sodium acetates clearly indicate that these were on the biosynthetic route from 1 to bisorbibutenolide (bislongiquinolide) 3 and bisorbicillinolide 4 via 2 as a branching point in the fungus.     

Implication of tyramine in the biosynthesis of morphinan alkaloids in Papaver

Doubly-labeled [³H, ¹⁴C]tyrosines, [1-¹³C-]tyramine or [2-¹⁴C]tyramine, administered to the stems of intact Papaver somniferum L. plants, were found to be incorporated into the morphinan alkaloids of the plant with comparable efficiency. ³H/¹⁴C ratios of alkaloids from plants fed the tyrosines were consistent with an almost equal conversion of this amino acid into the tetrahydroisoquinoline (TIQ) and benzyl-derived segments. Nuclear magnetic resonance (NMR) analyses of morphine isolated after administration of [1-¹³C]tyramine demonstrated selective labeling of C-16 of the alkaloid, indicating the conversion of this amine primarily into the TIQ-derived moiety. Morphine and thebaine labeled by [2-¹⁴C]tyramine were degraded to phenanthridines and N,N-dimethyl ethylamines. Of the total radioactivity in the alkaloids 97% was found to be associated with the ethylamines, a distribution consistent with the NMR data. This preferential utilization of tyramine in the biosynthesis of morphinan alkaloids can be explained by the compartmentalization of intermediates and enzymes of the pathway.Abbreviations L-dopa L-3,4-dihydroxyphenylalanine - HPLC high-pressure liquid chromatography - NMR nuelear magnetic resonance - TIQ tetrahydroisoquinoline     

Application of 13C-[2] - and 13C-[1,2] acetate in metabolic labelling studies of yeast and insect cells

The advantage of using ¹³C-labelled glucose in metabolic studies is that it is an important carbon and energy source for almost all biotechnologically and medically important organisms. On the other hand, the disadvantage is its relatively high cost in the labelling experiments. Looking for cheaper alternatives we found that ¹³C-[2] acetate or ¹³C-[1,2] acetate is a prospective compound for such experiments. Acetate is well incorporated by many organisms, including mammalian and insect cell cultures as preferred source of acetyl-CoA. Our experimental results using ¹³C NMR demonstrated that acetate was efficiently incorporated into glutamate and alanine secreted by the insect cell culture. Using D-stat culture of Saccharomyces uvarum on glucose/¹³C-acetate mineral media we demonstrated that the labelling patterns of proteinogenic amino acids can be well predicted on the basis of specific substrate consumption rates using the modified scheme of yeast metabolism and stoichiometric modelling. According to this scheme aspartate and alanine in S. uvarum under the experimental conditions used is synthesised in the mitochondria. Synthesis of alanine in the mitochondria was also demonstrated for Spodoptera frugiperda. For both organisms malic enzyme was also operative. For S. uvarum it was shown that the activity of malic enzyme is sufficient for supporting the mitochondrial biosynthetic reactions with NADPH.     

δ-Aminolaevulinate biosynthesis in the cyanobacterium Synechococcus 6301

The cyanobacterium (blue-green alga) Synechococcus 6301 incorporated a large amount of isotope from [1-¹⁴C] and [2-¹⁴C]acetate into phaeophorbide a obtained from chlorophyll a and into glutamatein cell protein; very little radioactivity was present in aspartate in cell protein. This distribution of isotope indicates that aspartate and the tetrapyrrole of chlorophyll a are not derived from a common C₄, precursor. The ratios of the specific radioactivities of phaeophorbide a to glutamate for organisms grown in the presence of 1-¹⁴C] and [2-⁴C ] acetate were 2.5:1 and 10:1 respectively. These are close to the theoretical values for the C₅, route to δ-aminolaevulinate which indicates that this is the only pathway to the tetrapyrrole precursor in Synechococcus 6301.     

Pathways of starch and sucrose biosynthesis in developing tubers of potato (Solanum tuberosum L.) and seeds of faba bean (Vicia faba L.)

Tissue slices from developing potato tubers (Solanum tuberosum L.) and developing cotyledons of faba bean (Vicia faba L.) were incubated with specifically labelled [¹³C]glucose and [¹³C]ribose. Enriched[¹³C]glucose released from starch granules was analysed by nuclear magnetic resonance (NMR). Spectral analyses were also performed on sucrose purified by high-performance liquid chromatography. In both tissues a low degree of randomisation (< 11 % in potato and < 14% in Vicia) was observed between carbon positions 1 and 6 in glucose released from starch when material was incubated with [¹³C]glucose labelled in positions 6 and 1, respectively. Similarly, with [2-¹³C]glucose a low degree of randomisation was observed in position 5. These findings indicate that extensive transport of three-carbon compounds across the amyloplast membrane does not occur in storage organs of either species. This is in agreement with previously published data which indicates that sixcarbon compounds are transported into the plastids during active starch synthesis. When [1-¹³C]ribose was used as a substrate, ¹³C-NMR spectra of starch indicated the operation of a classical pentose-phosphate pathway. However, with [2-¹³C]glucose there was no preferential enrichment in either carbon positions 1 or 3 relative to 4 or 6 of sucrose and starch (glucose). This provides evidence that entry of glucose in this pathway may be restricted in vivo. In both faba bean and potato the distribution of isotope between glucosyl and fructosyl moieties of sucrose approximated 50%. The degree of randomisation within glucosyl and fructosyl moieties ranged between 11 and 19.5%, indicating extensive recycling of triose phosphates.Abbreviation NMR nuclear magnetic resonance We are grateful to Dr. George Ratcliffe for his critical reading of the text and Dr. Bernard Goodman for helpful suggestions on the NMR measurements. The research was funded by a European Economic Community research grant, which the authors duly acknowledge.     

Biosynthesis of Protoheme and Heme a Precursors Solely from Glutamate in the Unicellular Red Alga Cyanidium caldarium

Two biosynthetic routes to the heme, chlorophyll, and phycobilin precursor, δ-aminolevulinic acid (ALA) are known: conversion of the intact five-carbon skeleton of glutamate, and ALA synthase-catalyzed condensation of glycine plus succinyl-coenzyme A. The existence and physiological roles of the two pathways in Cyanidium caldarium were assessed in vivo by determining the relative abilities of [2-¹⁴C]glycine and [1-¹⁴C]glutamate to label protoheme and heme a. Glutamate was incorporated to a much greater extent than glycine into both protoheme and heme a, even in cells that were unable to form chlorophyll and phycobilins. The small incorporation of glycine could be accounted for by transfer of label to intracellular glutamate pools, as determined from amino acid analysis. It thus appears that C. caldarium makes all tetrapyrroles, including mitochondrial hemes, solely from glutamate, and there is no contribution by ALA synthase in this organism.     

CARBON SOURCE DEPENDENCE OF THE RATIO OF δ‐AMINOLEVULINIC ACID BIOSYNTHESIS VIA THE C5 AND SHEMIN PATHWAYS IN EUGLENA GRACILIS (EUGLENOPHYCEAE)1

The ratio of two biosynthetic pathways was estimated, the C5 and Shemin pathways, to δ‐aminolevulinic acid (ALA, a biosynthetic intermediate of tetrapyrrole) from the ¹³C‐enrichment ratios (¹³C‐ER) at the carbon atoms of chl a (after conversion to methyl pheophorbide a) biosynthesized by Euglena gracilis G. A. Klebs when l ‐[3‐¹³C]alanine was used as a carbon source. On the basis of these estimations, we confirmed that ALA was efficiently biosynthesized via both the C5 and Shemin pathways in the plastids of E. gracilis, and we determined that the ratio of ALA biosynthesis via the Shemin pathway was increased in the ratio of 14%–67%, compared with that in our previous d ‐[1‐¹³C]glucose feeding experiment ( Iida et al. 2002 ). This carbon source dependence of the contributions of the two biosynthetic pathways might be related to activation of gluconeogenesis by the amino acid substrate. The methoxy carbon of the methoxycarbonyl group at C‐13² of chl a was labeled with the ¹³C‐carbon of l ‐[methyl‐¹³C]methionine derived from l ‐[3‐¹³C]alanine via [2‐¹³C]acetyl coenzyme A (CoA), through the atypical tricarboxylic acid (TCA) cycle, gluconeogenesis, and l‐ [3‐¹³C]serine. The phytyl moiety of chl a was also labeled on C‐P2, C‐P3¹, C‐P4, C‐P6, C‐P7¹, C‐P8, C‐P10, C‐P11¹, C‐P12, C‐P14, C‐P15¹, and C‐P16 from ¹³C‐isoprene (2‐[1,2‐methyl,3‐¹³C₃]methyl‐1,3‐butadiene) generated from l ‐[3‐¹³C]alanine via [2‐¹³C]acetyl CoA.     

Total synthesis of acetyl coenzyme a involved in autotrophic CO2 fixation inAcetobacterium woodii

The Gram positive anaerobeAcetobacterium woodii is able to grow autotrophically with a mixture of H₂ and CO₂ as the energy and carbon source. The question, by which pathway CO₂ is assimilated, was studied using long term isotope labeling.Autotrophically growing cultures produced acetate parallel to cell proliferation, and, when U-[¹⁴C]acetate was present as tracer, incorporated radioactivity into all cell fractions. The specific radioactivity and the label positions were determined for those representative cell compounds which biosynthetically originated directly from acetyl CoA (N-acetyl groups), pyruvate (alanine), oxaloacetate (aspartate), -ketoglutarate (glutamate), and hexosephosphates (glucosamine). Per mol compound the same amount of labeled acetate was incorporated into N-acetyl groups, alanine (C-2, C-3), aspartate (C-2, C-3), and twice the amount into glutamate (C-2, C-3, C-4, C-5) and into glucosamine. Consequently, the unlabeled carbon atoms of the C₃–C₆ compounds must have been derived from CO₂ by carboxylation subsequent to acetyl CoA synthesis. When 0.2 mM 2-[¹⁴C]pyruvate was added to autotrophically growing cultures, also a substantial amount of radioactivity was incorporated. Two important differences in comparison to the acetate experiment were observed: The N-acetyl groups were almost unlabeled and glutamate contained the same specific radioactivity as alanine or aspartate.These data showed that acetyl CoA is the central intermediate for biosynthesis and excluded the operation of the Calvin cycle inA. woodii. The results were consistent with the operation of a different autotrophic CO₂ fixation pathway in which CO₂ is converted into acetyl CoA by total synthesis via methyltetrahydrofolate; acetyl CoA is then further reductively carboxylated to pyruvate.     

Kreb's TCA cycle in Halobacterium salinarum investigated by 13C nuclear magnetic resonance spectroscopy

Kreb's tricarboxylic (TCA) cycle was studied in Halobacterium salinarum cells grown in the presence of glucose or alanine. The cells were incubated with ¹³C-labeled substrate and the labeling pattern of various carbon positions in glutamate was monitored by ¹³C-NMR spectroscopy. [2-¹³C]pyruvate, when used as a substrate, led mainly to signals for C-1 and C-5 glutamate, with some C-3 glutamate. [3-¹³C]pyruvate as a substrate produced signals, mainly C-2, C-3, and C-4 glutamate, with some C-1 and C-5 glutamate. The multiplicity of the signals and observation of a C-1 signal in this case indicates extensive cycling of the label in the TCA cycle. Isotopomer analysis of glutamate labeling suggested that of the total pyruvate entering the TCA cycle, the flux through pyruvate:ferredoxin oxidoreductase was 90% while that through pyruvate caboxylase was 10%. Only 53% of the total acetyl-CoA was produced from the added labeled pyruvate, the rest being generated endogenously. In the presence of nitrogen, mainly transamination reaction products were formed in the case of both these substrates. Received: November 26, 1997 / Accepted: May 11, 1998     

13C NMR evidence for bacteriochlorophyll c formation by the C5 pathway in green sulfur bacterium, Prosthecochloris

The 13C NMR spectra of the pheophorbide of bacteriochlorophyll c, formed in the presence of L-[1-13C]glutamate and [2-13C]glycine and [13C]bicarbonate in Prosthecochloris aestaurii, were analysed. The isotope in the glutamate was specifically incorporated into the eight carbon atoms in the tetrapyrrole macrocycle derived from the C-5 of 5-aminolevulinic acid, while no specific enrichment of these eight carbon atoms was observed in the spectrum of the pigment formed in the presence of [2-13C]glycine. These labelling patterns provide evidence for the operation of the C5 pathway of 5-aminolevulinic acid synthesis for bacteriochlorophyll c formation in the bacterium. The labelling of bacteriochlorophyll c by [13C]bicarbonate is consistent with its formation from 5-[1,4,5-13C]aminolevulinic acid formed by the C5 pathway from [1,2,5-13C]glutamic acid. It is proposed that this glutamate is the transamination product of 2-[1,2,5-13C]oxoglutaric acid, arising by carboxylation of [1,4-13C]succinyl-CoA with 13CO2 catalysed by 2-oxoglutaric acid synthase activity, and that the labelled succinyl-CoA is, in turn, derived by the fixation of 13CO2 by the reductive tricarboxylic acid cycle. The 13C chemical shifts of two sp2 quaternary carbons of bacteriopheophorbide c methyl ester (C-2 and C-4) were reassigned.     

Biosynthesis of vitamin B12

Radioactivity from [1-¹⁴C]riboflavin was incorporated into the 5,6-dimethylbenzimidazole moiety of Vitamin B₁₂ in the aerobes Bacillus megaterium, Nocardia rugosa and Streptomyces sp. as well as in the aerotolerant anaerobe Propionibacterium freudenreichii, but not in the anaerobe Eubacterium limosum.As recently published for E. limosum, also in the anaerobe Clostridium barkeri radioactivity from [1-¹⁴C]glycine and [2-¹⁴C]glycine was found in the 5,6-dimethylbenzimidazole moiety, but not in the corrin moiety. The addition of l-[methyl-¹⁴C]methionine to C. barkeri led to the labeling of the corrin moiety and the 5,6-dimethylbenzimidazole moiety, showing that the seven extra methyl groups in the corrin ring as well as the two methyl groups of the base part originate from this precursor.In Clostridium thermoaceticum, forming the vitamin B₁₂ analog 5-methoxybenzimidazolylcobamide, [1-¹⁴C]glycine and [2-¹⁴C]glycine were also incorporated into the 5-methoxybenzimidazole moiety, but not into the corrin ring.In E. limosum l-[U-¹⁴C]glutamate led to the labeling of the corrin ring of vitamin B₁₂, but not of its base moiety.There results together with data from the literature indicate that a common biosynthetic pathway might exist for the corrinoid biosynthesis in aerobic microorganisms, and in those aerotolerant anaerobes like the Propionibacteria, which form the 5,6-dimethylbenzimidazole moiety of vitamin B₁₂ only under aerobic conditions. They also show that this pathway differs from the pathway found in anaerobic bacteria.     

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     

6-Ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone from Hendersonula toruloidea: A biosynthetic study using C labels detected by nuclear magnetic resonance and C tracers

Production of 6-ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone was obtained by growth of Hendersonula toruloidea on Czapek-Dox broth supplemented with malt extract. Stationary cultures were grown at 28°C for 21–22 days yielding about 6 mg of metabolite per 700 ml of culture fluid. The best incorporations of isotopic tracers were obtained by addition at the 20th day of growth, followed by harvest 24–48 hr later. With [2-¹⁴C]acetate, incorporation values were in the range of 0.1–0.3% with dilution values from 2000 to 5900. With [1-¹⁴C]propionate, incorporations were much lower (0.04%) and dilutions much higher (120,000). Activity from [¹⁴CH₃]methionine was incorporated only into the OCH₃ groups (incorporation values, 0.5–0.7%). Nuclear magnetic resonance studies confirmed that propionate was not a precursor. Using [1,2-¹³C]acetate, substantial enrichments were obtained at all carbon atoms except those of the OCH₃ groups. The following pairs of carbon atoms were shown to be derived from acetate units: C-1 + 2, C-3 + 4, C-5 + 10, C-6 + 7, C-8 + 9, C-11 + 12. The biosynthetic pathway is clearly that of acetate plus polymalonate. Experiments with [2-¹³C²H₃]acetate suggested that the “starter” acetate unit was located at positions C-12 + 11.     

Characterization of S-layers from mesophilic bacillaceae and studies on their protective role towards muramidases

High resolution ¹³C NMR combined with chemical analysis were used to study the formation of metabolites from [1-¹³C]-labelled glucose by the salt-tolerant yeast Debaryomyces hansenii after transfer to media containing 8% NaCl. Time course spectroscopy of an aerobic cell suspension showed [1,3-¹³C]glycerol as the predominant end product. Perchloric acid extracts revealed additional less prominent incorporation of label into arabinitol, trehalose, glutamic acid, and alanine. The incorporation into trehalose and arabinitol showed a transient increase after shift to the high salinity medium. It is concluded that glycerol and arabinitol are the major organic solutes in D. hansenii, the production of glycerol being strongly induced by high salinity. Analysis of labelled extracts of D. hansenii after transfer to 8% NaCl media containing [1-¹³C]- or [6-¹³C]glucose, demonstrated that glucose is dissimilated via a combination of the Embden-Meyerhof-Parnas pathway and the pentose phosphate pathway, with the former playing a major role in glycerol formation and the latter in arabinitol production. The almost exclusive labelling of C5 of arabinitol from [6-¹³C]glucose indicates that the pathway to arabinitol proceeds via reduction of ribulose-5-phosphate.Abbreviations used NMR nuclear magnetic resonance - EMP Emden-Meyerhof-Parnas - PP pentose phosphate - GAP glyceraldehyde phosphate - DHAP dihydroxyacetone phosphate - ppm parts per million