[55] Assay of assimilatory enzymes in crude extracts of serine pathway methylotrophs

Goodwin, Patricia M.

doi:10.1016/0076-6879(90)88057-h

Cited by 18 publications

(12 citation statements)

References 6 publications

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“…Measurements were done at 30°C in a total volume of 0.9 ml. Malate synthase was measured in the direction of malate synthesis as described previously (24).…”

Section: Methodsmentioning

confidence: 99%

Alternative Route for Glyoxylate Consumption during Growth on Two-Carbon Compounds by Methylobacterium extorquens AM1

Okubo¹,

Yang²,

Chistoserdova³

et al. 2010

J Bacteriol

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Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and multicarbon compounds. Mutants defective in a pathway involved in converting acetyl-coenzyme A (CoA) to glyoxylate (the ethylmalonyl-CoA pathway) are unable to grow on both C 1 and C 2 compounds, showing that both modes of growth have this pathway in common. However, growth on C 2 compounds via the ethylmalonylCoA pathway should require glyoxylate consumption via malate synthase, but a mutant lacking malyl-CoA/ ␤-methylmalyl-CoA lyase activity (MclA1) that is assumed to be responsible for malate synthase activity still grows on C 2 compounds. Since glyoxylate is toxic to this bacterium, it seemed likely that a system is in place to keep it from accumulating. In this study, we have addressed this question and have shown by microarray analysis, mutant analysis, metabolite measurements, and 13 C-labeling experiments that M. extorquens AM1 contains an additional malyl-CoA/␤-methylmalyl-CoA lyase (MclA2) that appears to take part in glyoxylate metabolism during growth on C 2 compounds. In addition, an alternative pathway appears to be responsible for consuming part of the glyoxylate, converting it to glycine, methylene-H 4 F, and serine. Mutants lacking either pathway have a partial defect for growth on ethylamine, while mutants lacking both pathways are unable to grow appreciably on ethylamine. Our results suggest that the malate synthase reaction is a bottleneck for growth on C 2 compounds by this bacterium, which is partially alleviated by this alternative route for glyoxylate consumption. This strategy of multiple enzymes/pathways for the consumption of a toxic intermediate reflects the metabolic versatility of this facultative methylotroph and is a model for other metabolic networks involving high flux through toxic intermediates.Methylobacterium extorquens AM1 grows on one-carbon (C 1 ) compounds using the serine cycle for assimilation (25). This metabolism requires the conversion of acetyl-coenzyme A (CoA) to glyoxylate, which occurs via a novel pathway in which acetyl-CoA is converted to methylsuccinyl-CoA via acetoacetyl-CoA, ß-hydroxybutyryl-CoA, and ethylmalonyl-CoA (30-33). Recently, the steps involved in the conversion of methylsuccinyl-CoA to glyoxylate have been elucidated, and the pathway has been termed the ethylmalonyl-CoA (EMC) pathway (1,19,20,40). Careful labeling measurements coupled to measurements of intermediates has confirmed that, during the growth of M. extorquens AM1 on methanol, methylsuccinyl-CoA is converted to glyoxylate and propionyl-CoA via mesaconyl-CoA and ß-methylmalyl-CoA (40).This finding has raised questions regarding how M. extorquens AM1 grows on two-carbon (C 2 ) compounds. The pathway involved in the conversion of acetyl-CoA to glyoxylate is known to operate during growth on both C 1 and C 2 compounds, as mutants in genes involved in this conversion are unable to grow on either C 1 or C 2 compounds, and in both cases they are rescued by glyoxylate (11,(15)(16)(17)44). ...

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“…Measurements were done at 30°C in a total volume of 0.9 ml. Malate synthase was measured in the direction of malate synthesis as described previously (24).…”

Section: Methodsmentioning

confidence: 99%

Alternative Route for Glyoxylate Consumption during Growth on Two-Carbon Compounds by Methylobacterium extorquens AM1

Okubo¹,

Yang²,

Chistoserdova³

et al. 2010

J Bacteriol

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“…Hydroxypyruvate reductase was assayed as described by Chistoserdova & Lidstrom (1991). Malyl-CoA lyase activity was followed by measurement of acetyl-CoA disappearance (Goodwin, 1990). Methanol dehydrogenase, methylamine dehydrogenase, catechol dioxygenase and chloramphenicol acetyltransferase were assayed, respectively, as described by Anthony & Zatman (1964), Eady & Large (1968), Kataeva & Golovleva (1990) and Shaw (1975).…”

Section: Methodsmentioning

confidence: 99%

Molecular and mutational analysis of a DNA region separating two methylotrophy gene clusters in Methylobacterium extorquens AM1

Chistoserdova

Lidstrom

1997

Microbiology

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A region of 14.2 kb has been analysed that is a part of a locus on the Methylobacterium extorquens AM1 chromosome containing a number of Engineering of folate metabolism, and polypeptides of 13 and 20-5 kDa with no homologues in the protein database. Ten insertion mutations have been generated in the region to determine if the newly identified genes are associated with C, metabolism. A mutation in rnc/A, encoding malyl-CoA lyase, resulted in a C,-minus phenotype, while mutations in the other genes all showed a C,-plus phenotype. It was not possible to obtain null mutants in a putative folate metabolism gene, W C , implying the necessity of these folate synthesis genes for metabolism of C, and multicarbon compounds. Mutations in the putative ABC transporter genes, the genes similar to m a G and mxal, and other unidentified ORFs produced double-crossover recombinants with a C,-positive phenotype. Promoter regions have been investigated upstream of Orf3 and orf4 using the promoter probe vector pHX2OO. Transcription from these promoters was weak in wild-type M. extorquens AM1 but increased in regulatory MOX mutants.98195-1750, USA 1

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“…99. 8), methylamine dehydrogenase (EC 1 .4.99.3) and formaldehyde dehydrogenase (EC 1 .2.99.3) were assayed polarographically (Tatra & Goodwin, 1985;Dawson etal., 1990;Fordetal., 1985 Goodwin, 1990) ; glycerate kinase (EC 2.7.1 .31; Goodwin, 1990) ; serine : glyoxylate aminotransferase (EC 2.6.1 .45 ; Goodwin, 1990) ; acetyl-CoA independent phosphoenolpyruvate carboxylase (Goodwin, 1990 Mutants defective in the conversion of acetyl-CoA to glyoxylate F-proA2 recAl3 rpsL20 (Str') hsdS2O (hsdR hsdM) thipro hsdR recA; contains the tra region of RP4 integrated in the chromosome F-proA2 recA 1 endA 1 hsdR 17 (rim:) supE44 thi-1 recA 1 (lac) F' proAB lacPZAM15 F thrl ara 13 leu-6 axi8 tonA2 lacy1 minA 1 ginU44 gal6 minB2 rpsll35 supE44 lacUl69 (#80/acZAM15) hsdR 17 recA 1 endA 1 g r A 9 6 tbil relA 1…”

Section: Isolation Of Mutantsmentioning

confidence: 99%

A protein having similarity with methylmalonyl-CoA mutase is required for the assimilation of methanol and ethanol by Methylobacterium extorquens AM1

et al. 1996

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A 4.0 kb region of Methylobacterium extorquens AM1 DNA which complements three mutants unable to convert acetyl-CoA to glyoxylate (and therefore defective in the assimilation of methanol and ethanol) has been isolated and sequenced. It contains two ORFs and the 3'-end of a third one. The mutations in all three mutants mapped within the first ORF, which was designated meaA ; it encodes a protein having similarity with methylmalonylCoA mutase. However, methylmalonyl-CoA mutase was measured in extracts of one of the mutants and the specific activity was found to be similar to that in extracts of wild-type cells. Furthermore, although the predicted meaA gene product has the proposed cobalamin-binding site, it does not contain a highly conserved sequence (RIARNT) which is present in all known methylmalonylCoA mutases; meaA may therefore encode a novel vitamin-B,,-dependent enzyme. The predicted polypeptide encoded by the second ORF did not have similarity with any known proteins. The partial ORF encoded a protein with similarity with the 3-oxoacyl-[acyl-carrier-protein] reductases; it was not essential for growth on methanol or ethanol.

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[55] Assay of assimilatory enzymes in crude extracts of serine pathway methylotrophs

Cited by 18 publications

References 6 publications

Alternative Route for Glyoxylate Consumption during Growth on Two-Carbon Compounds by Methylobacterium extorquens AM1

Alternative Route for Glyoxylate Consumption during Growth on Two-Carbon Compounds by Methylobacterium extorquens AM1

Molecular and mutational analysis of a DNA region separating two methylotrophy gene clusters in Methylobacterium extorquens AM1

A protein having similarity with methylmalonyl-CoA mutase is required for the assimilation of methanol and ethanol by Methylobacterium extorquens AM1

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