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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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Methanethiol release and DXP formation are linked in R. rubrum(A) Time-dependent formation of intermediates in the MTA-isoprenoid shunt upon MTA-feeding. Cell suspensions of R. rubrum (1 ml, OD578=6.0) were incubated with 0.4 mM MTA and analyzed after 2, 10, and 20 minutes respectively by LC-FTMS-metabolomics. (B) Time dependent formation of free thiols by R. rubrum upon MTA uptake. Cell suspensions of R. rubrum (1 ml, OD578=4.0) were incubated with 0.4 mM MTA. The supernatant was analyzed for consumption of MTA and formation of free thiols. Free thiols formed were identified as methanethiol by HPLC and FTMS (Supplementary Fig. 4). At least two independent cell batches were used in these assays. Data represent mean values ± standard deviation. (C) LC-FTMS-Metabolomics analysis of MTA-isoprenoid shunt mutants. Cell suspensions of R. rubrum wild type and different mutants were incubated with MTA (according to A) and analyzed after 10 minutes by LC-FTMS-metabolomics, see Supplementary Fig. 6 for the detailed analysis of the cupin mutant (D) Thiol release activities by R. rubrum wild type and different mutants. Cell suspensions of R. rubrum were incubated with MTA according to (B), and the formation of free thiols over time was quantified. At least two independent cell batches were used in these assays. Data represent mean value ± standard deviation.
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Figure 3: Methanethiol release and DXP formation are linked in R. rubrum(A) Time-dependent formation of intermediates in the MTA-isoprenoid shunt upon MTA-feeding. Cell suspensions of R. rubrum (1 ml, OD578=6.0) were incubated with 0.4 mM MTA and analyzed after 2, 10, and 20 minutes respectively by LC-FTMS-metabolomics. (B) Time dependent formation of free thiols by R. rubrum upon MTA uptake. Cell suspensions of R. rubrum (1 ml, OD578=4.0) were incubated with 0.4 mM MTA. The supernatant was analyzed for consumption of MTA and formation of free thiols. Free thiols formed were identified as methanethiol by HPLC and FTMS (Supplementary Fig. 4). At least two independent cell batches were used in these assays. Data represent mean values ± standard deviation. (C) LC-FTMS-Metabolomics analysis of MTA-isoprenoid shunt mutants. Cell suspensions of R. rubrum wild type and different mutants were incubated with MTA (according to A) and analyzed after 10 minutes by LC-FTMS-metabolomics, see Supplementary Fig. 6 for the detailed analysis of the cupin mutant (D) Thiol release activities by R. rubrum wild type and different mutants. Cell suspensions of R. rubrum were incubated with MTA according to (B), and the formation of free thiols over time was quantified. At least two independent cell batches were used in these assays. Data represent mean value ± standard deviation.

Mentions: To identify subsequent reactions steps in MTA metabolism by R. rubrum we developed a liquid-chromatography Fourier transform mass spectrometry (LC-FTMS)-based metabolomics platform that would allow a comprehensive analysis of the R. rubrum metabolome upon perturbation with MTA. This platform combines high-resolution mass spectrometry with new semi-automated data analysis tools for the customized, convenient and highly confident assignment of metabolites based on molecular formula determination (see Methods). To find metabolites specifically affected by MTA-perturbation, suspensions of R. rubrum cells actively growing on minimal medium with sulfate as sole sulfur source were shortly deprived in sulfur-free medium (10 min), before they were transferred onto minimal medium with or without MTA as sole sulfur source and incubated for 10, and 20 min, respectively. Then, metabolites were extracted from whole cells and analyzed by the LC-FTMS platform, as shown in Supplementary Figure 2. Of the 1,406 individual peaks observed from extracts of wild type R. rubrum, we identified a total of 25 metabolites (1.8%) that were significantly up-regulated in MTA-fed cells (Supplementary Table 1). In agreement with the in extracto-NMR results, three of the peaks unique to MTA-feeding could be assigned to MTA, MTR-1P, and MTRu-1P, respectively, and were experimentally confirmed by spiking cell extracts with authentic standards (Supplementary Fig. 3). An additional peak (hypoxanthine) could be assigned as an intermediate in bacterial purine metabolism, which is well in line with downstream metabolism of adenine following its release from MTA upon MTR-1P formation. However, much to our surprise three peaks were significantly up-regulated upon feeding of MTA that could be assigned as metabolites of isoprenoid biosynthesis: 1-deoxyxylulose-5-phosphate (DXP), 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol (CDP-ME), and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (c-MEPP). Spiking of cell extracts with standards confirmed these assignments, indicating an unexpected intertwining of MTA metabolism and isoprenoid biosynthesis in R. rubrum (Supplementary Fig. 2). Realizing structural similarities between MTRu-1P and DXP, we hypothesized that DXP is a downstream metabolite of MTA, which is formed by release of the volatile methanethiol (CH3SH) from an intermediate in the pathway. Indeed, DXP formation in R. rubrum increased in a time dependent manner following MTA-feeding (Fig. 3a) as did the release of methanethiol in supernatants of R. rubrum cell suspensions (quantified with 5,5′-dithiobis(-2-nitrobenzoic acid, DTNB17, Fig. 3b). The rate of methanethiol release from MTA-fed cells (4 nmol min−1 OD578−1) was independent of the sulfur source used to grow R. rubrum (Supplementary Fig. 4), again indicating that MTA metabolism is a housekeeping function in this organism. Finally, when testing a RLP disruption strain13, we could neither observe an increase in DXP formation nor in methanethiol release, which confirmed the functional link between MTA metabolism and isoprenoid biosynthesis (Fig. 3c, 3d).


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)

Methanethiol release and DXP formation are linked in R. rubrum(A) Time-dependent formation of intermediates in the MTA-isoprenoid shunt upon MTA-feeding. Cell suspensions of R. rubrum (1 ml, OD578=6.0) were incubated with 0.4 mM MTA and analyzed after 2, 10, and 20 minutes respectively by LC-FTMS-metabolomics. (B) Time dependent formation of free thiols by R. rubrum upon MTA uptake. Cell suspensions of R. rubrum (1 ml, OD578=4.0) were incubated with 0.4 mM MTA. The supernatant was analyzed for consumption of MTA and formation of free thiols. Free thiols formed were identified as methanethiol by HPLC and FTMS (Supplementary Fig. 4). At least two independent cell batches were used in these assays. Data represent mean values ± standard deviation. (C) LC-FTMS-Metabolomics analysis of MTA-isoprenoid shunt mutants. Cell suspensions of R. rubrum wild type and different mutants were incubated with MTA (according to A) and analyzed after 10 minutes by LC-FTMS-metabolomics, see Supplementary Fig. 6 for the detailed analysis of the cupin mutant (D) Thiol release activities by R. rubrum wild type and different mutants. Cell suspensions of R. rubrum were incubated with MTA according to (B), and the formation of free thiols over time was quantified. At least two independent cell batches were used in these assays. Data represent mean value ± standard deviation.
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Figure 3: Methanethiol release and DXP formation are linked in R. rubrum(A) Time-dependent formation of intermediates in the MTA-isoprenoid shunt upon MTA-feeding. Cell suspensions of R. rubrum (1 ml, OD578=6.0) were incubated with 0.4 mM MTA and analyzed after 2, 10, and 20 minutes respectively by LC-FTMS-metabolomics. (B) Time dependent formation of free thiols by R. rubrum upon MTA uptake. Cell suspensions of R. rubrum (1 ml, OD578=4.0) were incubated with 0.4 mM MTA. The supernatant was analyzed for consumption of MTA and formation of free thiols. Free thiols formed were identified as methanethiol by HPLC and FTMS (Supplementary Fig. 4). At least two independent cell batches were used in these assays. Data represent mean values ± standard deviation. (C) LC-FTMS-Metabolomics analysis of MTA-isoprenoid shunt mutants. Cell suspensions of R. rubrum wild type and different mutants were incubated with MTA (according to A) and analyzed after 10 minutes by LC-FTMS-metabolomics, see Supplementary Fig. 6 for the detailed analysis of the cupin mutant (D) Thiol release activities by R. rubrum wild type and different mutants. Cell suspensions of R. rubrum were incubated with MTA according to (B), and the formation of free thiols over time was quantified. At least two independent cell batches were used in these assays. Data represent mean value ± standard deviation.
Mentions: To identify subsequent reactions steps in MTA metabolism by R. rubrum we developed a liquid-chromatography Fourier transform mass spectrometry (LC-FTMS)-based metabolomics platform that would allow a comprehensive analysis of the R. rubrum metabolome upon perturbation with MTA. This platform combines high-resolution mass spectrometry with new semi-automated data analysis tools for the customized, convenient and highly confident assignment of metabolites based on molecular formula determination (see Methods). To find metabolites specifically affected by MTA-perturbation, suspensions of R. rubrum cells actively growing on minimal medium with sulfate as sole sulfur source were shortly deprived in sulfur-free medium (10 min), before they were transferred onto minimal medium with or without MTA as sole sulfur source and incubated for 10, and 20 min, respectively. Then, metabolites were extracted from whole cells and analyzed by the LC-FTMS platform, as shown in Supplementary Figure 2. Of the 1,406 individual peaks observed from extracts of wild type R. rubrum, we identified a total of 25 metabolites (1.8%) that were significantly up-regulated in MTA-fed cells (Supplementary Table 1). In agreement with the in extracto-NMR results, three of the peaks unique to MTA-feeding could be assigned to MTA, MTR-1P, and MTRu-1P, respectively, and were experimentally confirmed by spiking cell extracts with authentic standards (Supplementary Fig. 3). An additional peak (hypoxanthine) could be assigned as an intermediate in bacterial purine metabolism, which is well in line with downstream metabolism of adenine following its release from MTA upon MTR-1P formation. However, much to our surprise three peaks were significantly up-regulated upon feeding of MTA that could be assigned as metabolites of isoprenoid biosynthesis: 1-deoxyxylulose-5-phosphate (DXP), 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol (CDP-ME), and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (c-MEPP). Spiking of cell extracts with standards confirmed these assignments, indicating an unexpected intertwining of MTA metabolism and isoprenoid biosynthesis in R. rubrum (Supplementary Fig. 2). Realizing structural similarities between MTRu-1P and DXP, we hypothesized that DXP is a downstream metabolite of MTA, which is formed by release of the volatile methanethiol (CH3SH) from an intermediate in the pathway. Indeed, DXP formation in R. rubrum increased in a time dependent manner following MTA-feeding (Fig. 3a) as did the release of methanethiol in supernatants of R. rubrum cell suspensions (quantified with 5,5′-dithiobis(-2-nitrobenzoic acid, DTNB17, Fig. 3b). The rate of methanethiol release from MTA-fed cells (4 nmol min−1 OD578−1) was independent of the sulfur source used to grow R. rubrum (Supplementary Fig. 4), again indicating that MTA metabolism is a housekeeping function in this organism. Finally, when testing a RLP disruption strain13, we could neither observe an increase in DXP formation nor in methanethiol release, which confirmed the functional link between MTA metabolism and isoprenoid biosynthesis (Fig. 3c, 3d).

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