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Crystal structure of Escherichia coli polynucleotide phosphorylase core bound to RNase E, RNA and manganese: implications for catalytic mechanism and RNA degradosome assembly.

Nurmohamed S, Vaidialingam B, Callaghan AJ, Luisi BF - J. Mol. Biol. (2009)

Bottom Line: At the centre of the PNPase ring is a tapered channel with an adjustable aperture where RNA bases stack on phenylalanine side chains and trigger structural changes that propagate to the active sites.Manganese can substitute for magnesium as an essential co-factor for PNPase catalysis, and our crystal structure of the enzyme in complex with manganese suggests how the metal is positioned to stabilise the transition state.We discuss the implications of these structural observations for the catalytic mechanism of PNPase, its processive mode of action, and its assembly into the RNA degradosome.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, UK.

ABSTRACT
Polynucleotide phosphorylase (PNPase) is a processive exoribonuclease that contributes to messenger RNA turnover and quality control of ribosomal RNA precursors in many bacterial species. In Escherichia coli, a proportion of the PNPase is recruited into a multi-enzyme assembly, known as the RNA degradosome, through an interaction with the scaffolding domain of the endoribonuclease RNase E. Here, we report crystal structures of E. coli PNPase complexed with the recognition site from RNase E and with manganese in the presence or in the absence of modified RNA. The homotrimeric PNPase engages RNase E on the periphery of its ring-like architecture through a pseudo-continuous anti-parallel beta-sheet. A similar interaction pattern occurs in the structurally homologous human exosome between the Rrp45 and Rrp46 subunits. At the centre of the PNPase ring is a tapered channel with an adjustable aperture where RNA bases stack on phenylalanine side chains and trigger structural changes that propagate to the active sites. Manganese can substitute for magnesium as an essential co-factor for PNPase catalysis, and our crystal structure of the enzyme in complex with manganese suggests how the metal is positioned to stabilise the transition state. We discuss the implications of these structural observations for the catalytic mechanism of PNPase, its processive mode of action, and its assembly into the RNA degradosome.

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Overlay of the protomers of E. coli PNPase core (grey) and S. antibioticus PNPase (purple; PDB code 1E3P). The S1 and KH domains, which are disordered, and are shown for the S. antibioticus structure.19,46 The manganese (green ball) is bound in the E. coli active site (inset).
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fig6: Overlay of the protomers of E. coli PNPase core (grey) and S. antibioticus PNPase (purple; PDB code 1E3P). The S1 and KH domains, which are disordered, and are shown for the S. antibioticus structure.19,46 The manganese (green ball) is bound in the E. coli active site (inset).

Mentions: Earlier, the active site of S. antibioticus PNPase was identified using the phosphate analogue tungstate, which was found to be coordinated by the side-chain and main-chain atoms of T462 and S463 (corresponding to E. coli PNPase residues S438 and S439; see insert of Fig. 6).19 The archaeal exosome has a phosphate at the corresponding location.24 Magnesium is required for PNPase enzymatic activity, and the metal is expected to be located in the vicinity of the phosphate-binding site. We find that Mn2+can substitute for Mg2+to support catalysis (S.N. et al., unpublished results), and since Mn2+can be more readily identified in difference maps and anomalous Fourier syntheses, we prepared RNA-free co-crystals of the PNPase core in the presence of 20 mM manganese acetate (Table 1). In this crystal form, a single protomer occupies the asymmetric unit, and the map revealed clear density for Mn2+at the active site. Mn2+is coordinated by the conserved residues D486, D492 and K494 (Figs. 2c and 6, inset), and it is likely that magnesium will bind in a similar manner. The metal-coordinating residues D486 and D492 may act in conjunction with the bound metal to support general acid/base catalysis. Consistent with the proposed metal-binding role of D492, substitution of the residue with glycine abolishes detectable phosphorolysis and polymerisation activities.7 These metal-coordinating residues are conserved also in human PNPase (Fig. 2c). The residues corresponding to D486, D492 and K494 are conserved also in RNase PH and the archaeal exosome, and they have been implicated in the catalytic mechanism of Bacillus subtilis and Aquifex aeolicus RNase PH21,22 and the Sulfolobus solfataricus exosome Rrp41 subunit.27 Consistent with its role in binding metal, the corresponding site was suggested to hold, at partial occupancy, a cadmium ion originating from the crystallisation buffer in the crystal structure of B. subtilis RNase PH.21 It seems likely that metal-assisted catalysis is conserved in archaeal exosomes, RNase PH and PNPase.


Crystal structure of Escherichia coli polynucleotide phosphorylase core bound to RNase E, RNA and manganese: implications for catalytic mechanism and RNA degradosome assembly.

Nurmohamed S, Vaidialingam B, Callaghan AJ, Luisi BF - J. Mol. Biol. (2009)

Overlay of the protomers of E. coli PNPase core (grey) and S. antibioticus PNPase (purple; PDB code 1E3P). The S1 and KH domains, which are disordered, and are shown for the S. antibioticus structure.19,46 The manganese (green ball) is bound in the E. coli active site (inset).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2723993&req=5

fig6: Overlay of the protomers of E. coli PNPase core (grey) and S. antibioticus PNPase (purple; PDB code 1E3P). The S1 and KH domains, which are disordered, and are shown for the S. antibioticus structure.19,46 The manganese (green ball) is bound in the E. coli active site (inset).
Mentions: Earlier, the active site of S. antibioticus PNPase was identified using the phosphate analogue tungstate, which was found to be coordinated by the side-chain and main-chain atoms of T462 and S463 (corresponding to E. coli PNPase residues S438 and S439; see insert of Fig. 6).19 The archaeal exosome has a phosphate at the corresponding location.24 Magnesium is required for PNPase enzymatic activity, and the metal is expected to be located in the vicinity of the phosphate-binding site. We find that Mn2+can substitute for Mg2+to support catalysis (S.N. et al., unpublished results), and since Mn2+can be more readily identified in difference maps and anomalous Fourier syntheses, we prepared RNA-free co-crystals of the PNPase core in the presence of 20 mM manganese acetate (Table 1). In this crystal form, a single protomer occupies the asymmetric unit, and the map revealed clear density for Mn2+at the active site. Mn2+is coordinated by the conserved residues D486, D492 and K494 (Figs. 2c and 6, inset), and it is likely that magnesium will bind in a similar manner. The metal-coordinating residues D486 and D492 may act in conjunction with the bound metal to support general acid/base catalysis. Consistent with the proposed metal-binding role of D492, substitution of the residue with glycine abolishes detectable phosphorolysis and polymerisation activities.7 These metal-coordinating residues are conserved also in human PNPase (Fig. 2c). The residues corresponding to D486, D492 and K494 are conserved also in RNase PH and the archaeal exosome, and they have been implicated in the catalytic mechanism of Bacillus subtilis and Aquifex aeolicus RNase PH21,22 and the Sulfolobus solfataricus exosome Rrp41 subunit.27 Consistent with its role in binding metal, the corresponding site was suggested to hold, at partial occupancy, a cadmium ion originating from the crystallisation buffer in the crystal structure of B. subtilis RNase PH.21 It seems likely that metal-assisted catalysis is conserved in archaeal exosomes, RNase PH and PNPase.

Bottom Line: At the centre of the PNPase ring is a tapered channel with an adjustable aperture where RNA bases stack on phenylalanine side chains and trigger structural changes that propagate to the active sites.Manganese can substitute for magnesium as an essential co-factor for PNPase catalysis, and our crystal structure of the enzyme in complex with manganese suggests how the metal is positioned to stabilise the transition state.We discuss the implications of these structural observations for the catalytic mechanism of PNPase, its processive mode of action, and its assembly into the RNA degradosome.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, UK.

ABSTRACT
Polynucleotide phosphorylase (PNPase) is a processive exoribonuclease that contributes to messenger RNA turnover and quality control of ribosomal RNA precursors in many bacterial species. In Escherichia coli, a proportion of the PNPase is recruited into a multi-enzyme assembly, known as the RNA degradosome, through an interaction with the scaffolding domain of the endoribonuclease RNase E. Here, we report crystal structures of E. coli PNPase complexed with the recognition site from RNase E and with manganese in the presence or in the absence of modified RNA. The homotrimeric PNPase engages RNase E on the periphery of its ring-like architecture through a pseudo-continuous anti-parallel beta-sheet. A similar interaction pattern occurs in the structurally homologous human exosome between the Rrp45 and Rrp46 subunits. At the centre of the PNPase ring is a tapered channel with an adjustable aperture where RNA bases stack on phenylalanine side chains and trigger structural changes that propagate to the active sites. Manganese can substitute for magnesium as an essential co-factor for PNPase catalysis, and our crystal structure of the enzyme in complex with manganese suggests how the metal is positioned to stabilise the transition state. We discuss the implications of these structural observations for the catalytic mechanism of PNPase, its processive mode of action, and its assembly into the RNA degradosome.

Show MeSH
Related in: MedlinePlus