Enzymes of 2-oxo acid degradation and biosynthesis in cell-free extracts of mixed rumen micro-organisms.
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ABSTRACT:
SUBMITTER: Bush RS
PROVIDER: S-EPMC1163857 | biostudies-other | 1976 Aug
REPOSITORIES: biostudies-other
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Project description:Mixed rumen micro-organisms, maintained in continuous culture readily incorporated labelled HCO3- and acetate into amino acids. Labelled propionate, in contrast, was utilized only for isoleucine biosynthesis, but failed to label other amino acids to any significant extent. Evidence was obtained showing that in these mixed, i.e. symbiotic, cultures foward tricarboxylic acid-cycle reactions only proceed to 2-oxoglutarate. 14C distribution in amino acids clearly shows that 2-oxoglutarate is not oxidized further by tricarboxylic acid-cycle enzymes. Instead, acetate is carboxylated to pyruvate which is then carboxylated to oxaloacetate. Oxaloacetate equilibrates with fumarate and thereby carbon atoms 1 and 4 as well as carbon atoms 2 and 3 are randomized. Evidence was also obtained for the carboxylation of propionate to 2-oxobutyrate, isovalerate to 4-methyl-2-oxopentanoate, phenylacetate and hydroxyphentlacetate to the corresponding phenyl- and hydroxyphenyl-pyruvic acids and succinate to 2-oxoglutarate. Of the amino acid precursors investigated, only 3-hydroxypyruvate, the precursor of serine, appeared to be synthesized via an oxidative step, i.e. 3-phosphoglyceric acid to 3-phosphohydroxypyruvic acid. Most 2-oxo precursors of amino acids in these organisms appear to be formed via reductive carboxylation of the precursor acid.
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Project description:1. alpha-[U-(14)C]Linolenic acid was incubated with the rumen contents of sheep and the metabolic products were characterized by thin-layer chromatography, gas-liquid chromatography and absorption spectroscopy in the ultraviolet and infrared. 2. A tentative scheme for the biohydrogenation route to stearic acid is presented. The main pathway is through diconjugated cis-cis-cis-octadecatrienoic acid, non-conjugated trans-cis (cis-trans)-octadecadienoic acid and trans-octadecenoic acid, but other pathways are apparent. 3. Washed rumen micro-organisms possessed only a limited capacity to hydrogenate alpha-linolenic acid and oleic acid but the rate was greatly stimulated by a factor(s) present in the supernatant rumen liquor. 4. Pure cultures of Clostridium perfringens, Streptococcus faecalis, Escherichia coli and a coliform organism isolated from sheep faeces possessed negligible ability to hydrogenate unsaturated fatty acids compared with a mixed population of rumen micro-organisms. Butyrivibrio fibrisolvens slowly converted linoleic acid into octadecenoic acid.
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Project description:Non-destructive DNA extraction from specimens and environmental samples using DESS preservation solution for DNA barcoding
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Project description:DNA metabarcoding focusing on the plankton community: an effective approach to reconstruct the paleo-environment
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Project description:Amino acid biosynthesis pathways observed in nature typically require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine: serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (CysK/CysM). To solve this chicken-and-egg problem, we substituted alternate amino acids in CysE, CysK and CysM for cysteine and methionine, which are the only two sulfur-containing proteinogenic amino acids. Using a cysteine-dependent auxotrophic E. coli strain, CysE function was rescued by cysteine-free and methionine-deficient enzymes, and CysM function was rescued by cysteine-free enzymes. CysK function, however, was not rescued in either case. Enzymatic assays showed that the enzymes responsible for rescuing the function in CysE and CysM also retained their activities in vitro. Additionally, substitution of the two highly conserved methionines in CysM decreased but did not eliminate overall activity. Engineering amino acid biosynthetic enzymes to lack the so-produced amino acids can provide insights into, and perhaps eventually fully recapitulate via a synthetic approach, the biogenesis of biotic amino acids.
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Project description:1. The action of cell-free filtrates from Trichoderma koningii was examined on undegraded cellulose in the form of cotton fibres, on degraded cellulose in the form of cellulose powder reprecipitated from phosphoric acid and on the soluble cellulose derivative CM-cellulose. 2. The cell-free filtrates compare favourably with intact cellulolytic micro-organisms in producing complete solubilization of undegraded as well as of degraded types of cellulose. Enzymic solubilization of cotton fibres gives quantitative conversion into glucose. Cellobiase is present. 3. The early enzymic breakdown of cotton fibres is characterized by the formation of very short fibres that increase to a maximum and disappear gradually by conversion into glucose. Disintegration of cotton fibres to short fibres is assisted by shaking, and within 20hr. the enzyme converts a minor fraction (up to 16%) of substrate into soluble products and a major portion (80%) into insoluble short fibres. 4. Maximum enzymic activity on cotton fibres occurs at about pH5.0, measured by the formation of short fibres, and at about pH3.8 on reprecipitated cellulose, measured by solubilization of the substrate. 5. Gluconolactone, glucose and cellobiose fail to produce marked inhibition of the enzymic hydrolysis of cotton fibres to short fibres or of the solubilization of reprecipitated cellulose unless present in amounts comparable with or greater than the initial weight of these two forms of cellulose. 6. The heavy-metal ions Cu(2+), Hg(2+) and Fe(3+) at 2mm concentration give 80-100% decrease in the enzymic breakdown of cotton fibres, measured by the formation of short fibres. At the same concentration Hg(2+) and Fe(3+) but not Cu(2+) also produce 70-100% inhibition of the solubilization of reprecipitated cellulose. 7. The ability to hydrolyse cotton fibres to short fibres and CM-cellulose to sugars is completely lost after heating enzyme preparations for 10min. at 71 degrees .
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Project description:Since skeletal muscle is the major site in the body for oxidation of leucine, isoleucine and valine, the pathway and control of leucine oxidation were investigated in cell-free preparations of rat muscle. Leucine was found to be transaminated to 4-methyl-2-oxopentanoate, which was then oxidatively decarboxylated. On differential centrifugation 70--80% of the transaminase activity was recovered in the soluble fraction of the cell, and the remaining amount in the mitochondrial fraction. The transaminase, from both fractions had similar pH optima and both were markedly inhibited by Ca2+. Thus changes in cellular Ca2+ concentration may regulate transaminase activity. Both transaminases had a much higher affinity for 2-oxoglutarate than for pyruvate. Therefore the utilization of amino groups from leucine for the biosynthesis of alanine in muscle [Odessey, Khairallah & Goldberg (1974) J. Biol. Chem. 249, 7623--7629] in vivo involves transamination with 2-oxoglutarate to produce glutamate, which is then transaminated with pyruvate to produce alanine. The dehydrogenase activity assayed by the decarboxylation of methyl-2-oxo[1-14C]pentanoate was localized exclusively in the fraction containing mitochondria and required NAD+, CoA and thiamin pyrophosphate for optimal activity. Measurements of competitive inhibition suggested that the oxo acids of leucine, isoleucine and valine are all decarboxylated by the same enzyme. The enzyme activity was decreased by 90% upon freezing or sonication and was stimulated severalfold by Mg2+, K+ and phosphate ions. In addition, it was markedly inhibited by ATP, but not by non-metabolizable analogues. This observation suggests that splitting of ATP is required for inhibition. The oxidative decarboxylation of 4-methyl-2-oxopentanoate by the dehydrogenase appears to be the rate-limiting step for leucine oxidation in muscle homogenates and also in intact tissues. In fact, rat muscles incubated with [1-14C]leucine release 1-14C-labelled oxo acid into the medium at rates comparable with the rate of decarboxylation. Intact muscles also released the oxo acids of [1-14C]valine or [1-14C]isoleucine, but not of other amino acids. These findings suggest that muscle is the primary source of the branched-chain oxo acids found in the blood.
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Project description:Recent progress in our understanding of the structural and catalytic properties of molybdenum-containing enzymes in eukaryotes is reviewed, along with aspects of the biosynthesis of the cofactor and its insertion into apoprotein.
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Project description:This study was carried out to assess the in vitro antioxidant, anti-inflammatory and antidiabetic effects of Nauclea latifolia (Sm.) leaf extracts. Ethanolic (NLE) and aqueous (NLA) extract of N. latifolia leaves were prepared and assessed for their anti-inflammatory activity, antioxidant potential, ?-amylase and ?-glucosidase inhibitory activities, and the mechanism of enzyme inhibition in vitro using standard established methods. From the results, phytochemicals such as flavonoids, phenolics, glycosides, and tannins were detected in both extracts of N. latifolia with NLE having a significantly (p < 0.05) higher phytochemical content. NLE displayed significantly (p < 0.05) better total antioxidant capacity, reducing power, 2,2-diphenyl-2-picrylhydrazyl, and hydrogen peroxide radical scavenging activities. For anti-inflammatory activities, 70.54 ± 2.45% albumin denaturation inhibition was observed for NLE while 68.05 ± 1.03% was recorded for NLA. Likewise, 16.07 ± 1.60 and 14.08 ± 1.76% were obtained against hypotonic solution and heat-induced erythrocyte haemolysis, respectively, for NLE while 20.59 ± 4.60 and 24.07 ± 1.60% were respective NLA values. NLE (IC50: 4.20 ± 0.18 and 1.19 ± 0.11?mg/mL) and NLA (IC50: 11.21 ± 0.35 and 2.64 ± 0.48?mg/mL) ?-glucosidase and ?-amylase inhibitory activities were dose-dependent with uncompetitive and competitive inhibition elicited, respectively, by the extracts. A significant positive association (p < 0.01 and 0.05) was identified between antioxidant activity and carbohydrate-metabolising enzyme inhibitory activity. The obtained result suggests N. latifolia leaf could serve as an alternative candidate for managing diabetes mellitus due to its antioxidant and anti-inflammatory association with diabetes-linked enzymes.
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Project description:The goal of bottom-up synthetic biology culminates in the assembly of an entire cell from separate biological building blocks. One major challenge resides in the in vitro production and implementation of complex genetic and metabolic pathways that can support essential cellular functions. Here, we show that phospholipid biosynthesis, a multiple-step process involved in cell membrane homeostasis, can be reconstituted starting from the genes encoding for all necessary proteins. A total of eight E. coli enzymes for acyl transfer and headgroup modifications were produced in a cell-free gene expression system and were co-translationally reconstituted in liposomes. Acyl-coenzyme A and glycerol-3-phosphate were used as canonical precursors to generate a variety of important bacterial lipids. Moreover, this study demonstrates that two-step acyl transfer can occur from enzymes synthesized inside vesicles. Besides clear implications for growth and potentially division of a synthetic cell, we postulate that gene-based lipid biosynthesis can become instrumental for ex vivo and protein purification-free production of natural and non-natural lipids.
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