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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

Phylogeny and function of RLPs(A) Phylogenetic tree of the RubisCO/RLP superfamily. The unrooted phylogenetic tree is based on amino acid sequence analysis of 333 representative proteins (listed in Supplementary Table 6) that were restricted to a length of 409 amino acids and aligned with ClustalW. Tree topography and evolutionary distance are given by the neighbor joining method. Numbers at nodes represent the percentage bootstrap values for the clades of this group in 500 replications. Similar trees were obtained by using the minimum evolution and the maximum likelihood method. For extended views of each subtree, see Supplementary Fig. 12 and 13. The scale bar represents a difference of 0.1 substitutions per site. RubisCOs are classified into three well established subfamilies2,29 (I, II and III). RLPs fall into six different subfamilies as described previously2,29: IV-AMC (metagenomic Leptospirillum sequences from an acid mine consortium); IV-DeepYkr; (R. rubrum group, including mainly alpha-, and gammaproteobacteria, some thermophilic species and Veillonellaceae); IV-YkrW (B. subtilis group, including many Bacilliales, Acidithiobacillales, and cyanobacteria); IV-GOS (metagenomic sequences from the global ocean sequencing program); IV-Photo (C. tepidum group, including many Chlorobiales and alphaproteobacteria,) and IV-NonPhoto (including many alpha-, and some beta proteobacteria). A seventh subgroup of RLPs, established in this extended phylogenetic analysis (IV-Aful, including Clostridiales and Archaeoglobus fulgidus) was described previously as a singleton (A. fulgidus DSM 4304) 2. (B) Function of the B. subtilis RLP in the classical methionine salvage pathway. Abbreviations: MTR, methylthioribose; DKMTP-1P, 2,3-diketo-5-methylthiopentyl-1-phosphate; HKMTP-1P, 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate; HKMTP 1,2-dihydroxy-3-keto-5-methylthiopentene; KMTB, 2,4-keto-4-methylthiobutyrate.
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Figure 1: Phylogeny and function of RLPs(A) Phylogenetic tree of the RubisCO/RLP superfamily. The unrooted phylogenetic tree is based on amino acid sequence analysis of 333 representative proteins (listed in Supplementary Table 6) that were restricted to a length of 409 amino acids and aligned with ClustalW. Tree topography and evolutionary distance are given by the neighbor joining method. Numbers at nodes represent the percentage bootstrap values for the clades of this group in 500 replications. Similar trees were obtained by using the minimum evolution and the maximum likelihood method. For extended views of each subtree, see Supplementary Fig. 12 and 13. The scale bar represents a difference of 0.1 substitutions per site. RubisCOs are classified into three well established subfamilies2,29 (I, II and III). RLPs fall into six different subfamilies as described previously2,29: IV-AMC (metagenomic Leptospirillum sequences from an acid mine consortium); IV-DeepYkr; (R. rubrum group, including mainly alpha-, and gammaproteobacteria, some thermophilic species and Veillonellaceae); IV-YkrW (B. subtilis group, including many Bacilliales, Acidithiobacillales, and cyanobacteria); IV-GOS (metagenomic sequences from the global ocean sequencing program); IV-Photo (C. tepidum group, including many Chlorobiales and alphaproteobacteria,) and IV-NonPhoto (including many alpha-, and some beta proteobacteria). A seventh subgroup of RLPs, established in this extended phylogenetic analysis (IV-Aful, including Clostridiales and Archaeoglobus fulgidus) was described previously as a singleton (A. fulgidus DSM 4304) 2. (B) Function of the B. subtilis RLP in the classical methionine salvage pathway. Abbreviations: MTR, methylthioribose; DKMTP-1P, 2,3-diketo-5-methylthiopentyl-1-phosphate; HKMTP-1P, 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate; HKMTP 1,2-dihydroxy-3-keto-5-methylthiopentene; KMTB, 2,4-keto-4-methylthiobutyrate.

Mentions: Given the importance of RubisCO in the global carbon cycle, much effort has been undertaken to understand the evolutionary relationship and functional diversity within the RubisCO-superfamily and its RLP members3–5. Based on phylogenetic analysis, active site differences and genome context, at least six different RLP-subfamilies were identified that are assumed to use different substrates and catalyze different reactions (Fig. 1a)2. However, correct functional assignments of RLPs are hampered by the fact that i) RubisCO itself is already an inefficient, naturally promiscuous enzyme6–8, and ii) potential substrate libraries of phosphorylated metabolites are not easily accessible. Although initial studies on the Chlorobium tepidum RLP-subfamily had established that RLPs might be involved in some aspect of sulfur metabolism3, the only group of RLPs for which an exact physiological function has been determined is the Bacillus subtilis RLP-subfamily4,9,10. RLPs in this group catalyze an enolization reaction in a bacterial variation of the ubiquitous “methionine salvage pathway” that converts 5′-methylthio-adenosine (MTA), a dead-end product of SAM-dependent polyamine biosynthesis, into L-methionine 4,11,12 (Fig. 1b).


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)

Phylogeny and function of RLPs(A) Phylogenetic tree of the RubisCO/RLP superfamily. The unrooted phylogenetic tree is based on amino acid sequence analysis of 333 representative proteins (listed in Supplementary Table 6) that were restricted to a length of 409 amino acids and aligned with ClustalW. Tree topography and evolutionary distance are given by the neighbor joining method. Numbers at nodes represent the percentage bootstrap values for the clades of this group in 500 replications. Similar trees were obtained by using the minimum evolution and the maximum likelihood method. For extended views of each subtree, see Supplementary Fig. 12 and 13. The scale bar represents a difference of 0.1 substitutions per site. RubisCOs are classified into three well established subfamilies2,29 (I, II and III). RLPs fall into six different subfamilies as described previously2,29: IV-AMC (metagenomic Leptospirillum sequences from an acid mine consortium); IV-DeepYkr; (R. rubrum group, including mainly alpha-, and gammaproteobacteria, some thermophilic species and Veillonellaceae); IV-YkrW (B. subtilis group, including many Bacilliales, Acidithiobacillales, and cyanobacteria); IV-GOS (metagenomic sequences from the global ocean sequencing program); IV-Photo (C. tepidum group, including many Chlorobiales and alphaproteobacteria,) and IV-NonPhoto (including many alpha-, and some beta proteobacteria). A seventh subgroup of RLPs, established in this extended phylogenetic analysis (IV-Aful, including Clostridiales and Archaeoglobus fulgidus) was described previously as a singleton (A. fulgidus DSM 4304) 2. (B) Function of the B. subtilis RLP in the classical methionine salvage pathway. Abbreviations: MTR, methylthioribose; DKMTP-1P, 2,3-diketo-5-methylthiopentyl-1-phosphate; HKMTP-1P, 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate; HKMTP 1,2-dihydroxy-3-keto-5-methylthiopentene; KMTB, 2,4-keto-4-methylthiobutyrate.
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Related In: Results  -  Collection

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Figure 1: Phylogeny and function of RLPs(A) Phylogenetic tree of the RubisCO/RLP superfamily. The unrooted phylogenetic tree is based on amino acid sequence analysis of 333 representative proteins (listed in Supplementary Table 6) that were restricted to a length of 409 amino acids and aligned with ClustalW. Tree topography and evolutionary distance are given by the neighbor joining method. Numbers at nodes represent the percentage bootstrap values for the clades of this group in 500 replications. Similar trees were obtained by using the minimum evolution and the maximum likelihood method. For extended views of each subtree, see Supplementary Fig. 12 and 13. The scale bar represents a difference of 0.1 substitutions per site. RubisCOs are classified into three well established subfamilies2,29 (I, II and III). RLPs fall into six different subfamilies as described previously2,29: IV-AMC (metagenomic Leptospirillum sequences from an acid mine consortium); IV-DeepYkr; (R. rubrum group, including mainly alpha-, and gammaproteobacteria, some thermophilic species and Veillonellaceae); IV-YkrW (B. subtilis group, including many Bacilliales, Acidithiobacillales, and cyanobacteria); IV-GOS (metagenomic sequences from the global ocean sequencing program); IV-Photo (C. tepidum group, including many Chlorobiales and alphaproteobacteria,) and IV-NonPhoto (including many alpha-, and some beta proteobacteria). A seventh subgroup of RLPs, established in this extended phylogenetic analysis (IV-Aful, including Clostridiales and Archaeoglobus fulgidus) was described previously as a singleton (A. fulgidus DSM 4304) 2. (B) Function of the B. subtilis RLP in the classical methionine salvage pathway. Abbreviations: MTR, methylthioribose; DKMTP-1P, 2,3-diketo-5-methylthiopentyl-1-phosphate; HKMTP-1P, 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate; HKMTP 1,2-dihydroxy-3-keto-5-methylthiopentene; KMTB, 2,4-keto-4-methylthiobutyrate.
Mentions: Given the importance of RubisCO in the global carbon cycle, much effort has been undertaken to understand the evolutionary relationship and functional diversity within the RubisCO-superfamily and its RLP members3–5. Based on phylogenetic analysis, active site differences and genome context, at least six different RLP-subfamilies were identified that are assumed to use different substrates and catalyze different reactions (Fig. 1a)2. However, correct functional assignments of RLPs are hampered by the fact that i) RubisCO itself is already an inefficient, naturally promiscuous enzyme6–8, and ii) potential substrate libraries of phosphorylated metabolites are not easily accessible. Although initial studies on the Chlorobium tepidum RLP-subfamily had established that RLPs might be involved in some aspect of sulfur metabolism3, the only group of RLPs for which an exact physiological function has been determined is the Bacillus subtilis RLP-subfamily4,9,10. RLPs in this group catalyze an enolization reaction in a bacterial variation of the ubiquitous “methionine salvage pathway” that converts 5′-methylthio-adenosine (MTA), a dead-end product of SAM-dependent polyamine biosynthesis, into L-methionine 4,11,12 (Fig. 1b).

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