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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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The proposed MTA-isoprenoid shunt in R. rubrumThe RLP is part of the central reaction sequence that involves the release of methanethiol from the molecule backbone. Whereas methanethiol can be recaptured as methionine via O-acetyl-L-homoserine sulfhydrylase (Rru_A0774), the rest of the molecule is converted into isoprenoid precursors. All intermediates of this proposed pathway that were identified by LC-FTMS-metabolomics are shown in boxed bar charts, with their individual increase in metabolite level after 0, 10 and 20 minutes feeding of MTA (+MTA, green bars) in comparison to control cells after 0, 10 and 20 min (control, black bars). Genes/proteins that were identified and characterized in this study are also shown and highlighted by colors.
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Figure 4: The proposed MTA-isoprenoid shunt in R. rubrumThe RLP is part of the central reaction sequence that involves the release of methanethiol from the molecule backbone. Whereas methanethiol can be recaptured as methionine via O-acetyl-L-homoserine sulfhydrylase (Rru_A0774), the rest of the molecule is converted into isoprenoid precursors. All intermediates of this proposed pathway that were identified by LC-FTMS-metabolomics are shown in boxed bar charts, with their individual increase in metabolite level after 0, 10 and 20 minutes feeding of MTA (+MTA, green bars) in comparison to control cells after 0, 10 and 20 min (control, black bars). Genes/proteins that were identified and characterized in this study are also shown and highlighted by colors.

Mentions: Next, we sought to identify genes important in MTA metabolism. By screening 10,200 transposon mutants of R. rubrum we identified four mutants in three genes that had lost the ability to release methanethiol upon MTA feeding (Supplementary Fig. 5), one of which was a mutant of the RLP gene (rru_A1998). The other two genes encode a putative MTA-phosphorylase (rru_A0361) and a putative MTR-1P isomerase (rru_A0360). Homologs of the three genes are conserved across the genomes of many bacterial species, where they are often organized as two distinct clusters (Supplementary Table 2). One cluster typically consists of the putative MTA-phosphorylase and the putative MTR-1P isomerase, whereas the second cluster comprises the RLP that is in many cases adjacent to a conserved gene encoding a small metal binding protein (cupin). Based on this observation, we consequently also created a cupin disruption strain. None of the mutant strains released methanethiol and consequently also did not show an increase in DXP-formation after MTA feeding, as demonstrated by LC-FTMS metabolomics (Fig. 3c, 3d). LC-FTMS metabolomic analysis enabled an even more detailed picture of each mutant that allowed functional assignments of the corresponding enzymes. The rru_A0361-mutant did not form MTR-1P or any other downstream metabolite, confirming its role as putative MTA-phosphorylase. MTA-metabolism in the rru_A0360-mutant stopped at the level of MTR-1P, according to its proposed role as MTR-1P-isomerase13. The rru_A1998 (rlp)-mutant accumulated MTRu-1P, as expected from the function of the RLP as an MTRu-1P isomerizing enzyme5. Finally, the cupin-mutant accumulated a mixture of MTRu-5P and MTXu-5P, affirming the role of RLP as MTRu-1P isomerase, and the cupin as being involved in processing these molecules (Fig. 3c, Supplementary Fig. 6). Based on these data, we propose the existence of a new bacterial pathway, the MTA-isoprenoid shunt that links SAM-dependent polyamine metabolism and isoprenoid biosynthesis, as summarized in Fig. 4. In this pathway, MTA, which is derived from polyamine biosynthesis, is first transformed into MTR-1P. MTR-1P is further isomerized to MTRu-1P, which then serves as substrate for the RLP. Due to the combined action of the RLP and the cupin protein, methanethiol is released, the phosphorylated ketose-coproduct is converted into DXP and further channeled into isoprenoid biosynthesis. Note that this proposed reaction sequence is also well supported by MTA-feeding metabolomics of R. rubrum wild type that showed the increase of all major metabolites of the proposed MTA-isoprenoid shunt over time, compared to (non-fed) control cells (Fig. 4).


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)

The proposed MTA-isoprenoid shunt in R. rubrumThe RLP is part of the central reaction sequence that involves the release of methanethiol from the molecule backbone. Whereas methanethiol can be recaptured as methionine via O-acetyl-L-homoserine sulfhydrylase (Rru_A0774), the rest of the molecule is converted into isoprenoid precursors. All intermediates of this proposed pathway that were identified by LC-FTMS-metabolomics are shown in boxed bar charts, with their individual increase in metabolite level after 0, 10 and 20 minutes feeding of MTA (+MTA, green bars) in comparison to control cells after 0, 10 and 20 min (control, black bars). Genes/proteins that were identified and characterized in this study are also shown and highlighted by colors.
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Related In: Results  -  Collection

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Figure 4: The proposed MTA-isoprenoid shunt in R. rubrumThe RLP is part of the central reaction sequence that involves the release of methanethiol from the molecule backbone. Whereas methanethiol can be recaptured as methionine via O-acetyl-L-homoserine sulfhydrylase (Rru_A0774), the rest of the molecule is converted into isoprenoid precursors. All intermediates of this proposed pathway that were identified by LC-FTMS-metabolomics are shown in boxed bar charts, with their individual increase in metabolite level after 0, 10 and 20 minutes feeding of MTA (+MTA, green bars) in comparison to control cells after 0, 10 and 20 min (control, black bars). Genes/proteins that were identified and characterized in this study are also shown and highlighted by colors.
Mentions: Next, we sought to identify genes important in MTA metabolism. By screening 10,200 transposon mutants of R. rubrum we identified four mutants in three genes that had lost the ability to release methanethiol upon MTA feeding (Supplementary Fig. 5), one of which was a mutant of the RLP gene (rru_A1998). The other two genes encode a putative MTA-phosphorylase (rru_A0361) and a putative MTR-1P isomerase (rru_A0360). Homologs of the three genes are conserved across the genomes of many bacterial species, where they are often organized as two distinct clusters (Supplementary Table 2). One cluster typically consists of the putative MTA-phosphorylase and the putative MTR-1P isomerase, whereas the second cluster comprises the RLP that is in many cases adjacent to a conserved gene encoding a small metal binding protein (cupin). Based on this observation, we consequently also created a cupin disruption strain. None of the mutant strains released methanethiol and consequently also did not show an increase in DXP-formation after MTA feeding, as demonstrated by LC-FTMS metabolomics (Fig. 3c, 3d). LC-FTMS metabolomic analysis enabled an even more detailed picture of each mutant that allowed functional assignments of the corresponding enzymes. The rru_A0361-mutant did not form MTR-1P or any other downstream metabolite, confirming its role as putative MTA-phosphorylase. MTA-metabolism in the rru_A0360-mutant stopped at the level of MTR-1P, according to its proposed role as MTR-1P-isomerase13. The rru_A1998 (rlp)-mutant accumulated MTRu-1P, as expected from the function of the RLP as an MTRu-1P isomerizing enzyme5. Finally, the cupin-mutant accumulated a mixture of MTRu-5P and MTXu-5P, affirming the role of RLP as MTRu-1P isomerase, and the cupin as being involved in processing these molecules (Fig. 3c, Supplementary Fig. 6). Based on these data, we propose the existence of a new bacterial pathway, the MTA-isoprenoid shunt that links SAM-dependent polyamine metabolism and isoprenoid biosynthesis, as summarized in Fig. 4. In this pathway, MTA, which is derived from polyamine biosynthesis, is first transformed into MTR-1P. MTR-1P is further isomerized to MTRu-1P, which then serves as substrate for the RLP. Due to the combined action of the RLP and the cupin protein, methanethiol is released, the phosphorylated ketose-coproduct is converted into DXP and further channeled into isoprenoid biosynthesis. Note that this proposed reaction sequence is also well supported by MTA-feeding metabolomics of R. rubrum wild type that showed the increase of all major metabolites of the proposed MTA-isoprenoid shunt over time, compared to (non-fed) control cells (Fig. 4).

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