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Identifying reaction modules in metabolic pathways: bioinformatic deduction and experimental validation of a new putative route in purine catabolism.

Barba M, Dutoit R, Legrain C, Labedan B - BMC Syst Biol (2013)

Bottom Line: Finally, we present experimental data supporting the conclusion that this UGTCase is likely to be involved in a new route in purine catabolism.It will help us to trace how the primordial promiscuous enzymes were assembled progressively in functional modules, as the present pathways diverged from ancestral pathways to give birth to the present-day mechanistically diversified superfamilies.In addition, the concept allows the determination of the actual function of misannotated proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut de Génétique et Microbiologie, CNRS UMR 8621, Université Paris Sud, Bâtiment 400, 91405, Orsay Cedex, France. bernard.labedan@igmors.u-psud.fr.

ABSTRACT

Background: Enzymes belonging to mechanistically diverse superfamilies often display similar catalytic mechanisms. We previously observed such an association in the case of the cyclic amidohydrolase superfamily whose members play a role in related steps of purine and pyrimidine metabolic pathways. To establish a possible link between enzyme homology and chemical similarity, we investigated further the neighbouring steps in the respective pathways.

Results: We identified that successive reactions of the purine and pyrimidine pathways display similar chemistry. These mechanistically-related reactions are often catalyzed by homologous enzymes. Detection of series of similar catalysis made by succeeding enzyme families suggested some modularity in the architecture of the central metabolism. Accordingly, we introduce the concept of a reaction module to define at least two successive steps catalyzed by homologous enzymes in pathways alignable by similar chemical reactions. Applying such a concept allowed us to propose new function for misannotated paralogues. In particular, we discovered a putative ureidoglycine carbamoyltransferase (UGTCase) activity. Finally, we present experimental data supporting the conclusion that this UGTCase is likely to be involved in a new route in purine catabolism.

Conclusions: Using the reaction module concept should be of great value. It will help us to trace how the primordial promiscuous enzymes were assembled progressively in functional modules, as the present pathways diverged from ancestral pathways to give birth to the present-day mechanistically diversified superfamilies. In addition, the concept allows the determination of the actual function of misannotated proteins.

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Analysis of the purified Rxyl_2847 enzyme. (A) SDS-PAGE of purified Rxyl_2847 enzyme on a NuPage Bis-Tris 4-12% gel (Life Technologies) in MOPS running buffer. Proteins were stained with PageRuler protein staining solution (ThermoScientific). Lane 1, PageRuler Unstained Broad Range Protein Ladder (ThermoScientific); Lane 2, 10 μg of purified Rxyl_2847 enzyme. (B) Western blot analysis of purified Rxyl_2847 enzyme (same condition of electrophoresis as in (A), electroblot onto Hybond (GE Healthcare) nitrocellulose membrane). Lane 1, PageRuler Unstained Broad Range Protein Ladder (ThermoScientific); Lane 2, 100 ng of purified Rxyl_2847 enzyme; Lane 3, 100 ng of a purified His-tagged protein control; Lane 4, 100 ng of a purified untagged protein control.
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Figure 8: Analysis of the purified Rxyl_2847 enzyme. (A) SDS-PAGE of purified Rxyl_2847 enzyme on a NuPage Bis-Tris 4-12% gel (Life Technologies) in MOPS running buffer. Proteins were stained with PageRuler protein staining solution (ThermoScientific). Lane 1, PageRuler Unstained Broad Range Protein Ladder (ThermoScientific); Lane 2, 10 μg of purified Rxyl_2847 enzyme. (B) Western blot analysis of purified Rxyl_2847 enzyme (same condition of electrophoresis as in (A), electroblot onto Hybond (GE Healthcare) nitrocellulose membrane). Lane 1, PageRuler Unstained Broad Range Protein Ladder (ThermoScientific); Lane 2, 100 ng of purified Rxyl_2847 enzyme; Lane 3, 100 ng of a purified His-tagged protein control; Lane 4, 100 ng of a purified untagged protein control.

Mentions: Recombinant His-tagged enzyme was purified to near-homogeneity by a three-step procedure including heat-treatment, metal affinity chromatography and molecular sieving (see Figure 8 and Methods). SDS-PAGE showed a subunit molecular mass of 37 kDa but also a major band at 80 kDa (Figure 8A). The western blot analysis of purified enzyme (Figure 8B) pointed out that it corresponds to a dimeric state of Rxyl_2847. Such phenomenon was already reported for other thermophilic enzymes [41,42]. The activity of the purified enzyme was examined in the physiological, catabolic direction, i.e. the phosphorolysis of allantoate. Since the equilibrium of the reaction catalyzed by carbamoyltransferases strongly favours the carbamoylation direction, in vitro studies of the catabolic reaction require the removal of one of the products formed. This can be achieved by using arsenate instead of phosphate [43] or by coupling the reaction in vivo to that of a carbamate kinase, or an anabolic carbamoyltransferase. In this work, the E. coli OTCase, purified as described previously [44] was used in the presence of ornithine to convert the carbamoyl phosphate produced by the phosphorolysis of allantoate to citrulline (Table 3).


Identifying reaction modules in metabolic pathways: bioinformatic deduction and experimental validation of a new putative route in purine catabolism.

Barba M, Dutoit R, Legrain C, Labedan B - BMC Syst Biol (2013)

Analysis of the purified Rxyl_2847 enzyme. (A) SDS-PAGE of purified Rxyl_2847 enzyme on a NuPage Bis-Tris 4-12% gel (Life Technologies) in MOPS running buffer. Proteins were stained with PageRuler protein staining solution (ThermoScientific). Lane 1, PageRuler Unstained Broad Range Protein Ladder (ThermoScientific); Lane 2, 10 μg of purified Rxyl_2847 enzyme. (B) Western blot analysis of purified Rxyl_2847 enzyme (same condition of electrophoresis as in (A), electroblot onto Hybond (GE Healthcare) nitrocellulose membrane). Lane 1, PageRuler Unstained Broad Range Protein Ladder (ThermoScientific); Lane 2, 100 ng of purified Rxyl_2847 enzyme; Lane 3, 100 ng of a purified His-tagged protein control; Lane 4, 100 ng of a purified untagged protein control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016543&req=5

Figure 8: Analysis of the purified Rxyl_2847 enzyme. (A) SDS-PAGE of purified Rxyl_2847 enzyme on a NuPage Bis-Tris 4-12% gel (Life Technologies) in MOPS running buffer. Proteins were stained with PageRuler protein staining solution (ThermoScientific). Lane 1, PageRuler Unstained Broad Range Protein Ladder (ThermoScientific); Lane 2, 10 μg of purified Rxyl_2847 enzyme. (B) Western blot analysis of purified Rxyl_2847 enzyme (same condition of electrophoresis as in (A), electroblot onto Hybond (GE Healthcare) nitrocellulose membrane). Lane 1, PageRuler Unstained Broad Range Protein Ladder (ThermoScientific); Lane 2, 100 ng of purified Rxyl_2847 enzyme; Lane 3, 100 ng of a purified His-tagged protein control; Lane 4, 100 ng of a purified untagged protein control.
Mentions: Recombinant His-tagged enzyme was purified to near-homogeneity by a three-step procedure including heat-treatment, metal affinity chromatography and molecular sieving (see Figure 8 and Methods). SDS-PAGE showed a subunit molecular mass of 37 kDa but also a major band at 80 kDa (Figure 8A). The western blot analysis of purified enzyme (Figure 8B) pointed out that it corresponds to a dimeric state of Rxyl_2847. Such phenomenon was already reported for other thermophilic enzymes [41,42]. The activity of the purified enzyme was examined in the physiological, catabolic direction, i.e. the phosphorolysis of allantoate. Since the equilibrium of the reaction catalyzed by carbamoyltransferases strongly favours the carbamoylation direction, in vitro studies of the catabolic reaction require the removal of one of the products formed. This can be achieved by using arsenate instead of phosphate [43] or by coupling the reaction in vivo to that of a carbamate kinase, or an anabolic carbamoyltransferase. In this work, the E. coli OTCase, purified as described previously [44] was used in the presence of ornithine to convert the carbamoyl phosphate produced by the phosphorolysis of allantoate to citrulline (Table 3).

Bottom Line: Finally, we present experimental data supporting the conclusion that this UGTCase is likely to be involved in a new route in purine catabolism.It will help us to trace how the primordial promiscuous enzymes were assembled progressively in functional modules, as the present pathways diverged from ancestral pathways to give birth to the present-day mechanistically diversified superfamilies.In addition, the concept allows the determination of the actual function of misannotated proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut de Génétique et Microbiologie, CNRS UMR 8621, Université Paris Sud, Bâtiment 400, 91405, Orsay Cedex, France. bernard.labedan@igmors.u-psud.fr.

ABSTRACT

Background: Enzymes belonging to mechanistically diverse superfamilies often display similar catalytic mechanisms. We previously observed such an association in the case of the cyclic amidohydrolase superfamily whose members play a role in related steps of purine and pyrimidine metabolic pathways. To establish a possible link between enzyme homology and chemical similarity, we investigated further the neighbouring steps in the respective pathways.

Results: We identified that successive reactions of the purine and pyrimidine pathways display similar chemistry. These mechanistically-related reactions are often catalyzed by homologous enzymes. Detection of series of similar catalysis made by succeeding enzyme families suggested some modularity in the architecture of the central metabolism. Accordingly, we introduce the concept of a reaction module to define at least two successive steps catalyzed by homologous enzymes in pathways alignable by similar chemical reactions. Applying such a concept allowed us to propose new function for misannotated paralogues. In particular, we discovered a putative ureidoglycine carbamoyltransferase (UGTCase) activity. Finally, we present experimental data supporting the conclusion that this UGTCase is likely to be involved in a new route in purine catabolism.

Conclusions: Using the reaction module concept should be of great value. It will help us to trace how the primordial promiscuous enzymes were assembled progressively in functional modules, as the present pathways diverged from ancestral pathways to give birth to the present-day mechanistically diversified superfamilies. In addition, the concept allows the determination of the actual function of misannotated proteins.

Show MeSH
Related in: MedlinePlus