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A RubisCO-like protein links SAM metabolism with isoprenoid biosynthesis.

Erb TJ, Evans BS, Cho K, Warlick BP, Sriram J, Wood BM, Imker HJ, Sweedler JV, Tabita FR, Gerlt JA - Nat. Chem. Biol. (2012)

Bottom Line: Functional assignment of uncharacterized proteins is a challenge in the era of large-scale genome sequencing.Here, we combine in extracto NMR, proteomics and transcriptomics with a newly developed (knock-out) metabolomics platform to determine a potential physiological role for a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-like protein from Rhodospirillum rubrum.Our studies unraveled an unexpected link in bacterial central carbon metabolism between S-adenosylmethionine-dependent polyamine metabolism and isoprenoid biosynthesis and also provide an alternative approach to assign enzyme function at the organismic level.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Champaign, IL, USA.

ABSTRACT
Functional assignment of uncharacterized proteins is a challenge in the era of large-scale genome sequencing. Here, we combine in extracto NMR, proteomics and transcriptomics with a newly developed (knock-out) metabolomics platform to determine a potential physiological role for a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-like protein from Rhodospirillum rubrum. Our studies unraveled an unexpected link in bacterial central carbon metabolism between S-adenosylmethionine-dependent polyamine metabolism and isoprenoid biosynthesis and also provide an alternative approach to assign enzyme function at the organismic level.

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Related in: MedlinePlus

Characterization of the initial steps in MTA-metabolism of R. rubrum by in-extracto NMR1H-NMR spectra of R. rubrum cell extract incubated with MTA that show the transformation of MTA into MTR-1P, and MTRu-1P over time. 0.78 mg cell extract protein of R. rubrum grown on MTA as sole sulfur source were incubated with 0.4 mM MTA at 30 °C. Spectra were recorded at different time points as indicated. Characteristic 1H-NMR signals for MTA (green), MTR-1P (purple), and MTRu-1P (blue) are indicated by colored lines. The full array experiment is shown in Supplementary Figure 1.
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Figure 2: Characterization of the initial steps in MTA-metabolism of R. rubrum by in-extracto NMR1H-NMR spectra of R. rubrum cell extract incubated with MTA that show the transformation of MTA into MTR-1P, and MTRu-1P over time. 0.78 mg cell extract protein of R. rubrum grown on MTA as sole sulfur source were incubated with 0.4 mM MTA at 30 °C. Spectra were recorded at different time points as indicated. Characteristic 1H-NMR signals for MTA (green), MTR-1P (purple), and MTRu-1P (blue) are indicated by colored lines. The full array experiment is shown in Supplementary Figure 1.

Mentions: In order to elucidate the unknown MTA metabolic pathway in R. rubrum, we first used 1H-NMR to trace the fate of MTA in R. rubrum cell extracts (in extracto-NMR). For that purpose, extracts of R. rubrum were prepared from cells actively growing with MTA as sole sulfur source (generation time td=16.7 ± 3.0 h). Then, these extracts were exchanged into buffered D2O, MTA was added and 1H-NMR spectra were continuously recorded over time (Supplementary Results, Supplementary Fig. 1). MTA was first converted into 5-methylthio-D-ribose-1-phosphate (MTR-1P) with the release of adenine. Subsequently, MTR-1P was isomerized into MTRu-1P (Fig. 2). The overall rate for this reaction sequence (0.7 + 0.3 nmol min−1 mg−1 protein) closely matched the theoretically expected minimal activity for MTA metabolic enzymes (0.6 nmol min−1 mg−1) to sustain the observed growth of R. rubrum, as calculated from the specific aerobic growth rate of R. rubrum on MTA (μ=0.042 + 0.008h−1, Supplementary Fig. 1). Notably, we could demonstrate MTRu-1P formation at comparable rates also in extracts of sulfate-grown cells, suggesting that MTA metabolism serves a housekeeping function in R. rubrum, analogous to the reactions of the classical methionine salvage pathway in B. subtilis14–16. However, in neither extract could metabolites downstream of MTRu-1P be detected by 1H-NMR, indicating that further investigations are limited by the in extracto NMR-method used.


A RubisCO-like protein links SAM metabolism with isoprenoid biosynthesis.

Erb TJ, Evans BS, Cho K, Warlick BP, Sriram J, Wood BM, Imker HJ, Sweedler JV, Tabita FR, Gerlt JA - Nat. Chem. Biol. (2012)

Characterization of the initial steps in MTA-metabolism of R. rubrum by in-extracto NMR1H-NMR spectra of R. rubrum cell extract incubated with MTA that show the transformation of MTA into MTR-1P, and MTRu-1P over time. 0.78 mg cell extract protein of R. rubrum grown on MTA as sole sulfur source were incubated with 0.4 mM MTA at 30 °C. Spectra were recorded at different time points as indicated. Characteristic 1H-NMR signals for MTA (green), MTR-1P (purple), and MTRu-1P (blue) are indicated by colored lines. The full array experiment is shown in Supplementary Figure 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475740&req=5

Figure 2: Characterization of the initial steps in MTA-metabolism of R. rubrum by in-extracto NMR1H-NMR spectra of R. rubrum cell extract incubated with MTA that show the transformation of MTA into MTR-1P, and MTRu-1P over time. 0.78 mg cell extract protein of R. rubrum grown on MTA as sole sulfur source were incubated with 0.4 mM MTA at 30 °C. Spectra were recorded at different time points as indicated. Characteristic 1H-NMR signals for MTA (green), MTR-1P (purple), and MTRu-1P (blue) are indicated by colored lines. The full array experiment is shown in Supplementary Figure 1.
Mentions: In order to elucidate the unknown MTA metabolic pathway in R. rubrum, we first used 1H-NMR to trace the fate of MTA in R. rubrum cell extracts (in extracto-NMR). For that purpose, extracts of R. rubrum were prepared from cells actively growing with MTA as sole sulfur source (generation time td=16.7 ± 3.0 h). Then, these extracts were exchanged into buffered D2O, MTA was added and 1H-NMR spectra were continuously recorded over time (Supplementary Results, Supplementary Fig. 1). MTA was first converted into 5-methylthio-D-ribose-1-phosphate (MTR-1P) with the release of adenine. Subsequently, MTR-1P was isomerized into MTRu-1P (Fig. 2). The overall rate for this reaction sequence (0.7 + 0.3 nmol min−1 mg−1 protein) closely matched the theoretically expected minimal activity for MTA metabolic enzymes (0.6 nmol min−1 mg−1) to sustain the observed growth of R. rubrum, as calculated from the specific aerobic growth rate of R. rubrum on MTA (μ=0.042 + 0.008h−1, Supplementary Fig. 1). Notably, we could demonstrate MTRu-1P formation at comparable rates also in extracts of sulfate-grown cells, suggesting that MTA metabolism serves a housekeeping function in R. rubrum, analogous to the reactions of the classical methionine salvage pathway in B. subtilis14–16. However, in neither extract could metabolites downstream of MTRu-1P be detected by 1H-NMR, indicating that further investigations are limited by the in extracto NMR-method used.

Bottom Line: Functional assignment of uncharacterized proteins is a challenge in the era of large-scale genome sequencing.Here, we combine in extracto NMR, proteomics and transcriptomics with a newly developed (knock-out) metabolomics platform to determine a potential physiological role for a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-like protein from Rhodospirillum rubrum.Our studies unraveled an unexpected link in bacterial central carbon metabolism between S-adenosylmethionine-dependent polyamine metabolism and isoprenoid biosynthesis and also provide an alternative approach to assign enzyme function at the organismic level.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Champaign, IL, USA.

ABSTRACT
Functional assignment of uncharacterized proteins is a challenge in the era of large-scale genome sequencing. Here, we combine in extracto NMR, proteomics and transcriptomics with a newly developed (knock-out) metabolomics platform to determine a potential physiological role for a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-like protein from Rhodospirillum rubrum. Our studies unraveled an unexpected link in bacterial central carbon metabolism between S-adenosylmethionine-dependent polyamine metabolism and isoprenoid biosynthesis and also provide an alternative approach to assign enzyme function at the organismic level.

Show MeSH
Related in: MedlinePlus