Salmonella typhimurium LT2 catabolizes propionate via the 2-methylcitric acid cycle - PubMed (original) (raw)

Salmonella typhimurium LT2 catabolizes propionate via the 2-methylcitric acid cycle

A R Horswill et al. J Bacteriol. 1999 Sep.

Abstract

We previously identified the prpBCDE operon, which encodes catabolic functions required for propionate catabolism in Salmonella typhimurium. Results from (13)C-labeling experiments have identified the route of propionate breakdown and determined the biochemical role of each Prp enzyme in this pathway. The identification of catabolites accumulating in wild-type and mutant strains was consistent with propionate breakdown through the 2-methylcitric acid cycle. Our experiments demonstrate that the alpha-carbon of propionate is oxidized to yield pyruvate. The reactions are catalyzed by propionyl coenzyme A (propionyl-CoA) synthetase (PrpE), 2-methylcitrate synthase (PrpC), 2-methylcitrate dehydratase (probably PrpD), 2-methylisocitrate hydratase (probably PrpD), and 2-methylisocitrate lyase (PrpB). In support of this conclusion, the PrpC enzyme was purified to homogeneity and shown to have 2-methylcitrate synthase activity in vitro. (1)H nuclear magnetic resonance spectroscopy and negative-ion electrospray ionization mass spectrometry identified 2-methylcitrate as the product of the PrpC reaction. Although PrpC could use acetyl-CoA as a substrate to synthesize citrate, kinetic analysis demonstrated that propionyl-CoA is the preferred substrate.

PubMed Disclaimer

Figures

FIG. 1

FIG. 1

Proposed propionate breakdown pathways in E. coli and S. typhimurium. Pathways: 1, α-hydroxyglutarate; 2, citramalate; 3, methylmalonyl-CoA; 4, acryloyl-CoA; 5, 2-methylcitric acid cycle.

FIG. 2

FIG. 2

Propionate catabolites visualized by 13C-NMR spectroscopy. Strain TR6583 (prpBCDE+) was grown in minimal medium supplemented with [2-13C]propionate to accumulate 13C-labeled propionate breakdown intermediates. (A) Proton-decoupled 13C-NMR spectrum of an extract from this culture. (B) Proton-coupled 13C-NMR spectrum of this extract. The boxed carbon atoms in the chemical structures above the peaks indicate the source of the carbon signals.

FIG. 3

FIG. 3

In vitro conversion of [2-13C]propionate to [2-13C]propionyl-CoA by PrpE. Dialyzed cell extracts of strain JE4184 were used in these experiments. (A) Proton-decoupled 13C-NMR spectrum of the reaction products. (B) Proton-coupled 13C-NMR spectrum of the reaction products.

FIG. 4

FIG. 4

Propionate catabolites that accumulate in a prpD mutant. Strain JE3914 (prpD174) was grown in minimal medium supplemented with [2-13C]propionate to accumulate 13C-labeled propionate breakdown intermediates. (A) Proton-decoupled spectrum of an extract from this culture. (B) Proton-coupled spectrum of this extract. The boxed carbon atoms in the chemical structures above the peaks indicate the source of the carbon signals.

FIG. 5

FIG. 5

Propionate catabolites that accumulate in a prpB mutant. Strain JE3946 (prpB195) was grown in minimal medium supplemented with [2-13C]propionate to accumulate 13C-labeled propionate breakdown intermediates. (A) Proton-decoupled spectrum of an extract from this culture. (B) Proton-coupled spectrum of this extract. The boxed carbon atoms in the chemical structures above the peaks indicate the source of the carbon signals.

FIG. 6

FIG. 6

Spectroscopic analysis of the PrpC reaction product. (A) 1H-NMR spectrum; (B) homodecoupling 1H-NMR spectrum with irradiation at the methyl doublet; (C) ESI mass spectrum. The structure shown is 2-methylcitrate.

FIG. 7

FIG. 7

SDS-PAGE analysis of homogeneous PrpC. Lanes: A, molecular mass standards (in decreasing mass order): phosphorylase b, bovine serum albumin, glutamate dehydrogenase, ovalbumin, aldolase, carbonic anhydrase, lysozyme; B, His-tagged PrpC (3 μg) (approximately 45-kDa monomer).

FIG. 8

FIG. 8

Pathway for propionate catabolism in S. typhimurium. The results presented in this paper support the breakdown of propionate via 2-methylcitrate. The reactions catalyzed by enzymes encoded in the prpBCDE operon are indicated. A possible route for oxaloacetate regeneration and gluconeogenesis is shown (pps, PEP synthetase; Ppc, PEP carboxylase).

FIG. 9

FIG. 9

Comparison of the prpBC intergenic region between S. typhimurium and E. coli. The 91-bp repeats and proposed ORF in E. coli are indicated.

Similar articles

Cited by

References

    1. Aoki H, Uchiyama H, Umetsu H, Tabuchi T. Isolation of 2-methylcitrate dehydratase, a new enzyme serving in the methylcitric acid cycle for propionate metabolism, from Yallowia lipolytica. Biosci Biotechnol Biochem. 1995;59:1825–1828.
    1. Ausubel F A, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K. Current protocols in molecular biology. New York, N.Y: Greene Publishing Associates & Wiley Interscience; 1989.
    1. Blair J M. Magnesium and aconitase equilibrium: determination of apparent stability constants of magnesium substrate complexes from equilibrium data. Eur J Biochem. 1969;8:287–291. - PubMed
    1. Blattner F R, Plunkett III G, Bloch C A, Perna N T, Burland V, Riley M, Collado-Vides J, Glasner J D, Rode C K, Mayhew G F, Gregor J, Davis N W, Kirkpatrick H A, Goeden M A, Rose D J, Mau B, Shao Y. The complete genome sequence of Escherichia coli K-12. Science. 1997;277:1453–1474. - PubMed
    1. Chan R K, Botstein D, Watanabe T, Ogata Y. Specialized transduction of tetracycline resistance by phage P22 in Salmonella typhimurium. II. Properties of a high transducing lysate. Virology. 1972;50:883–898. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources