Characterization of methylmalonyl-CoA mutase involved in the propionate photoassimilation of Euglena gracilis Z
Emi MiyamotoYuri TaniokaAyako Nishizawa‐YokoiYukinori YabutaKouhei OhnishiHaruo MisonoShigeru ShigeokaYoshihisa NakanoFumio Watanabe
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Euglena gracilis
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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.
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The photosynthetic protozoon Euglena gracilis Z contains adenosylcobalamin-dependent methylmalonyl-CoA mutase (MCM) involved in propionate metabolism. The specific activity of the Euglena mutase was about 6·5-fold greater in propionate-adapted Euglena cells than in photoautotrophic cells (control). Although the control cells contained only one mutase (apparent M r 72000), the propionate-adapted cells contained two mutases with M r values of 72000 and 17000; both enzymes were located in the mitochondria. These results provide evidence that propionate-adapted Euglena contains two MCM isozymes. The induced mutase (M r 17000) permits photoassimilation of propionate.
Euglena gracilis
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Euglena
Phosphoglycerate mutase
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Gluconeogenesis
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Euglena gracilis
Euglena
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ABSTRACT Phosphoglucosamine mutase (GlmM) catalyzes the formation of glucosamine-1-phosphate from glucosamine-6-phosphate, an essential step in the pathway for UDP- N -acetylglucosamine biosynthesis in bacteria. This enzyme must be phosphorylated to be active and acts according to a ping-pong mechanism involving glucosamine-1,6-diphosphate as an intermediate (L. Jolly, P. Ferrari, D. Blanot, J. van Heijenoort, F. Fassy, and D. Mengin-Lecreulx, Eur. J. Biochem. 262:202–210, 1999). However, the process by which the initial phosphorylation of the enzyme is achieved in vivo remains unknown. Here we show that the phosphoglucosamine mutase from Escherichia coli autophosphorylates in vitro in the presence of [ 32 P]ATP. The same is observed with phosphoglucosamine mutases from other bacterial species, yeast N -acetylglucosamine-phosphate mutase, and rabbit muscle phosphoglucomutase. Labeling of the E. coli GlmM enzyme with [ 32 P]ATP requires the presence of a divalent cation, and the label is subsequently lost when the enzyme is incubated with either of its substrates. Analysis of enzyme phosphorylation by high-pressure liquid chromatography and coupled mass spectrometry confirms that only one phosphate has been covalently linked to the enzyme. Only phosphoserine could be detected after acid hydrolysis of the labeled protein, and site-directed mutagenesis of serine residues located in or near the active site identifies the serine residue at position 102 as the site of autophosphorylation of E. coli GlmM.
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Two isozymes of chorismate mutase (CA mutase 1 and CA mutase 2 ) and two isozymes of prephenate dehydratase (PPA dehydratase 1 and PPA dehydratase 2 ) have been found in Pseudomonas aeruginosa . The activities CA mutase 2 -PPA dehydratase 2 catalyzing phenylalanine biosynthesis have been purified almost 40-fold and were found to be associated as a bifunctional enzyme or an enzyme complex. The enzymes specific for tyrosine biosynthesis did not appear to manifest such physical association. Thus, the organization of enzymes concerned with phenylalanine and tyrosine biosynthesis in P. aeruginosa is unique and is unlike most other organisms. Single site mutants have been isolated which have lost both CA mutase 2 -PPA dehydratase 2 activities resulting in a requirement for phenylalanine for growth. Single site revertants of these mutants regained both these activities simultaneously and were able to grow on minimal medium. A mutant, r 6 , was also isolated which had normal CA mutase 2 but lacked PPA dehydratase 2 activity.
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Phenylalanine hydroxylase
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ABSTRACT Euglena gracilis is a suitable model system to investigate the role of zinc in the process of cell division. In zinc-deficient organisms there is a characteristic arrest of cellular proliferation, the DNA content of the cells doubles, whereas RNA and protein contents decrease. The present investigations include the growth characteristics, changes in cellular morphology at various stages in the growth cycle, quantitation of zinc uptake and incorporation of tritium-labelled precursors into RNA by organisms grown in zinc sufficient (Zn+), (Zn,+ content 1 ×10−5 M) or zinc-deficient (Zn−), (Zn+ content 1 × 10−7 M) medium. Cell division ceases on depletion of zinc from the medium. There are 20-fold less cells in (Zn−) medium than in control cultures. The size of (Zn+) cells decreases during log phase due to a reduction in the paramylon content of the cytoplasm. The size of (Zn−) cells, however, increases, due to an accumulation of paramylon. This results in a 13-fold increment in dry weight compared to control. Other cytoplasmic organelles, including Golgi bodies, mitochondria, etc. are normal. Nuclear morphology also is unchanged. There is a reduction in the rate of incorporation of labelled precursors into RNA by (Zn−) cells. The DNA content of (Zn−) E. gracilis, the absence of morphologic evidence to indicate that cell division has followed the doubling of the DNA, and the arrest in proliferation suggests that a critical zinc-dependent step in the cell cycle, localized to G2, is blocked in zinc deficiency.
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Phosphohexomutase superfamily enzymes catalyze the reversible intramolecular transfer of a phosphoryl moiety on hexose sugars. Bacillus subtilis phosphoglucomutase PgcA catalyzes the reversible interconversion of glucose 6-phosphate (Glc-6-P) and glucose 1-phosphate (Glc-1-P), a precursor of UDP-glucose (UDP-Glc). B. subtilis phosphoglucosamine mutase (GlmM) is a member of the same enzyme superfamily that converts glucosamine 6-phosphate (GlcN-6-P) to glucosamine 1-phosphate (GlcN-1-P), a precursor of the amino sugar moiety of peptidoglycan. Here, we present evidence that B. subtilis PgcA possesses activity as a phosphoglucosamine mutase that contributes to peptidoglycan biosynthesis. This activity was made genetically apparent by the synthetic lethality of pgcA with glmR, a positive regulator of amino sugar biosynthesis, which can be specifically suppressed by overproduction of GlmM. A gain-of-function mutation in a substrate binding loop (PgcA G47S) increases this secondary activity and suppresses a glmR mutant. Our results demonstrate that bacterial phosphoglucomutases may possess secondary phosphoglucosamine mutase activity, and that this dual activity may provide some level of functional redundancy for the essential peptidoglycan biosynthesis pathway.
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Phosphoglucomutase
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