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Ribosomal oxygenases are structurally conserved from prokaryotes to humans.

Chowdhury R, Sekirnik R, Brissett NC, Krojer T, Ho CH, Ng SS, Clifton IJ, Ge W, Kershaw NJ, Fox GC, Muniz JR, Vollmar M, Phillips C, Pilka ES, Kavanagh KL, von Delft F, Oppermann U, McDonough MA, Doherty AJ, Schofield CJ - Nature (2014)

Bottom Line: Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone N(ε)-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery.The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts.This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.

View Article: PubMed Central - PubMed

Affiliation: The Department of Chemistry and Oxford Centre for Integrative Systems Biology, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK.

ABSTRACT
2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components and in the hydroxylation of transcription factors and splicing factor proteins. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA and ribosomal proteins have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone N(ε)-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.

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Features of ROX-substrate bindingRibbons representations of Mina53 (a), NO66 (b) and ycfDRM (c) monomers showing difference electron density (Fo-Fc OMIT) for substrates contoured to 3σ (right panels). Left panels show active site surface representations, showing key hydrogen-bonds/polar interactions (dotted lines) with substrates. a, With Mina53, the His39rpL27a imidazole nitrogens form hydrogen-bonds with Tyr167/Ser257 (NδHis39-OHTyr167 2.9 Å; NεHis39-OγSer257 3.1 Å). b, In NO66, His216rpL8 is similarly bound in a deep pocket; the His216rpL8 imidazole nitrogens form hydrogen-bonds with Tyr328/Ser421 (NδHis216-OHTyr328 3.2 Å; NεHis216-OγSer421 2.7 Å) and hydrophobic interactions with Ile244, that project its pro-S hydrogen toward the metal (metal-β-CH2, 4.4 Å). While Mina53 (a) uses 4 primary amides, Asn101, Gln136, Gln139 and Asn165 to interact with rpL27a backbone amides, NO66 (b) uses 2 arginines (272, 297) to hydrogen-bond with the Asn215rpL8 sidechain and His216rpL8 backbone. In the ycfDRM·L16 complex (c), Arg82L16 binds in a pocket defined by the Tyr137/Met120 sidechains, which form π-cation and hydrophobic interactions with Arg82L16 sidechain. The Arg82 guanidino group makes electrostatic interactions with the Asp118ycfDRM carboxylate (O-NH, 2.8-3.1 Å) and hydrogen-bonds to Ser208ycfDRM (NεArg82-OHSer208 3.5 Å; NηArg82-COSer208 3.2 Å). Although Tyr167Mina53/Tyr328NO66 are not positionally related to Tyr137ycfDRM, the role of the serine (Ser257Mina53, Ser421NO66, Ser208ycfDRM, β-VIII) in binding the hydroxylated His/Arg is conserved in ROX. Substitutions of these residues cause significant loss of activity (see Extended Data Fig. 6).
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Figure 3: Features of ROX-substrate bindingRibbons representations of Mina53 (a), NO66 (b) and ycfDRM (c) monomers showing difference electron density (Fo-Fc OMIT) for substrates contoured to 3σ (right panels). Left panels show active site surface representations, showing key hydrogen-bonds/polar interactions (dotted lines) with substrates. a, With Mina53, the His39rpL27a imidazole nitrogens form hydrogen-bonds with Tyr167/Ser257 (NδHis39-OHTyr167 2.9 Å; NεHis39-OγSer257 3.1 Å). b, In NO66, His216rpL8 is similarly bound in a deep pocket; the His216rpL8 imidazole nitrogens form hydrogen-bonds with Tyr328/Ser421 (NδHis216-OHTyr328 3.2 Å; NεHis216-OγSer421 2.7 Å) and hydrophobic interactions with Ile244, that project its pro-S hydrogen toward the metal (metal-β-CH2, 4.4 Å). While Mina53 (a) uses 4 primary amides, Asn101, Gln136, Gln139 and Asn165 to interact with rpL27a backbone amides, NO66 (b) uses 2 arginines (272, 297) to hydrogen-bond with the Asn215rpL8 sidechain and His216rpL8 backbone. In the ycfDRM·L16 complex (c), Arg82L16 binds in a pocket defined by the Tyr137/Met120 sidechains, which form π-cation and hydrophobic interactions with Arg82L16 sidechain. The Arg82 guanidino group makes electrostatic interactions with the Asp118ycfDRM carboxylate (O-NH, 2.8-3.1 Å) and hydrogen-bonds to Ser208ycfDRM (NεArg82-OHSer208 3.5 Å; NηArg82-COSer208 3.2 Å). Although Tyr167Mina53/Tyr328NO66 are not positionally related to Tyr137ycfDRM, the role of the serine (Ser257Mina53, Ser421NO66, Ser208ycfDRM, β-VIII) in binding the hydroxylated His/Arg is conserved in ROX. Substitutions of these residues cause significant loss of activity (see Extended Data Fig. 6).

Mentions: ROX structures were determined in complex with Mn(II) and 2OG/N-oxalylglycine (NOG), replacing Fe(II) and 2OG. As for most 2OG-oxygenases, the metal is octahedrally coordinated by a 2-His-1-carboxylate triad from DSBH-βII and - βVII14,15 (Fig. 3); 2 coordination sites are occupied by the 2OG/NOG oxalyl group leaving one for H2O/O2 binding (Fig. 4 and Extended Data Fig. 4). With the ycfDs the NOG C5-carboxylate is positioned to salt bridge with Arg140ycfD/Arg148ycfDRM on DSBH-βIV (Extended Data Fig. 4). This arrangement is notable because with other 2OG-oxygenases where the 2OG C5-carboxylate interacts with an Arg-residue, it is located on βVIII14,15. In hROX, the 2OG C5-carboxylate interacting residue is a lysine (Lys194Mina53/Lys355NO66) from βIV, as in most JmjC-hydroxylases and KDMs. These observations lead to the proposal that the eukaryotic JmjC-hydroxylases/KDMs evolved from prokaryotic ycfDs/ROX.


Ribosomal oxygenases are structurally conserved from prokaryotes to humans.

Chowdhury R, Sekirnik R, Brissett NC, Krojer T, Ho CH, Ng SS, Clifton IJ, Ge W, Kershaw NJ, Fox GC, Muniz JR, Vollmar M, Phillips C, Pilka ES, Kavanagh KL, von Delft F, Oppermann U, McDonough MA, Doherty AJ, Schofield CJ - Nature (2014)

Features of ROX-substrate bindingRibbons representations of Mina53 (a), NO66 (b) and ycfDRM (c) monomers showing difference electron density (Fo-Fc OMIT) for substrates contoured to 3σ (right panels). Left panels show active site surface representations, showing key hydrogen-bonds/polar interactions (dotted lines) with substrates. a, With Mina53, the His39rpL27a imidazole nitrogens form hydrogen-bonds with Tyr167/Ser257 (NδHis39-OHTyr167 2.9 Å; NεHis39-OγSer257 3.1 Å). b, In NO66, His216rpL8 is similarly bound in a deep pocket; the His216rpL8 imidazole nitrogens form hydrogen-bonds with Tyr328/Ser421 (NδHis216-OHTyr328 3.2 Å; NεHis216-OγSer421 2.7 Å) and hydrophobic interactions with Ile244, that project its pro-S hydrogen toward the metal (metal-β-CH2, 4.4 Å). While Mina53 (a) uses 4 primary amides, Asn101, Gln136, Gln139 and Asn165 to interact with rpL27a backbone amides, NO66 (b) uses 2 arginines (272, 297) to hydrogen-bond with the Asn215rpL8 sidechain and His216rpL8 backbone. In the ycfDRM·L16 complex (c), Arg82L16 binds in a pocket defined by the Tyr137/Met120 sidechains, which form π-cation and hydrophobic interactions with Arg82L16 sidechain. The Arg82 guanidino group makes electrostatic interactions with the Asp118ycfDRM carboxylate (O-NH, 2.8-3.1 Å) and hydrogen-bonds to Ser208ycfDRM (NεArg82-OHSer208 3.5 Å; NηArg82-COSer208 3.2 Å). Although Tyr167Mina53/Tyr328NO66 are not positionally related to Tyr137ycfDRM, the role of the serine (Ser257Mina53, Ser421NO66, Ser208ycfDRM, β-VIII) in binding the hydroxylated His/Arg is conserved in ROX. Substitutions of these residues cause significant loss of activity (see Extended Data Fig. 6).
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Figure 3: Features of ROX-substrate bindingRibbons representations of Mina53 (a), NO66 (b) and ycfDRM (c) monomers showing difference electron density (Fo-Fc OMIT) for substrates contoured to 3σ (right panels). Left panels show active site surface representations, showing key hydrogen-bonds/polar interactions (dotted lines) with substrates. a, With Mina53, the His39rpL27a imidazole nitrogens form hydrogen-bonds with Tyr167/Ser257 (NδHis39-OHTyr167 2.9 Å; NεHis39-OγSer257 3.1 Å). b, In NO66, His216rpL8 is similarly bound in a deep pocket; the His216rpL8 imidazole nitrogens form hydrogen-bonds with Tyr328/Ser421 (NδHis216-OHTyr328 3.2 Å; NεHis216-OγSer421 2.7 Å) and hydrophobic interactions with Ile244, that project its pro-S hydrogen toward the metal (metal-β-CH2, 4.4 Å). While Mina53 (a) uses 4 primary amides, Asn101, Gln136, Gln139 and Asn165 to interact with rpL27a backbone amides, NO66 (b) uses 2 arginines (272, 297) to hydrogen-bond with the Asn215rpL8 sidechain and His216rpL8 backbone. In the ycfDRM·L16 complex (c), Arg82L16 binds in a pocket defined by the Tyr137/Met120 sidechains, which form π-cation and hydrophobic interactions with Arg82L16 sidechain. The Arg82 guanidino group makes electrostatic interactions with the Asp118ycfDRM carboxylate (O-NH, 2.8-3.1 Å) and hydrogen-bonds to Ser208ycfDRM (NεArg82-OHSer208 3.5 Å; NηArg82-COSer208 3.2 Å). Although Tyr167Mina53/Tyr328NO66 are not positionally related to Tyr137ycfDRM, the role of the serine (Ser257Mina53, Ser421NO66, Ser208ycfDRM, β-VIII) in binding the hydroxylated His/Arg is conserved in ROX. Substitutions of these residues cause significant loss of activity (see Extended Data Fig. 6).
Mentions: ROX structures were determined in complex with Mn(II) and 2OG/N-oxalylglycine (NOG), replacing Fe(II) and 2OG. As for most 2OG-oxygenases, the metal is octahedrally coordinated by a 2-His-1-carboxylate triad from DSBH-βII and - βVII14,15 (Fig. 3); 2 coordination sites are occupied by the 2OG/NOG oxalyl group leaving one for H2O/O2 binding (Fig. 4 and Extended Data Fig. 4). With the ycfDs the NOG C5-carboxylate is positioned to salt bridge with Arg140ycfD/Arg148ycfDRM on DSBH-βIV (Extended Data Fig. 4). This arrangement is notable because with other 2OG-oxygenases where the 2OG C5-carboxylate interacts with an Arg-residue, it is located on βVIII14,15. In hROX, the 2OG C5-carboxylate interacting residue is a lysine (Lys194Mina53/Lys355NO66) from βIV, as in most JmjC-hydroxylases and KDMs. These observations lead to the proposal that the eukaryotic JmjC-hydroxylases/KDMs evolved from prokaryotic ycfDs/ROX.

Bottom Line: Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone N(ε)-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery.The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts.This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.

View Article: PubMed Central - PubMed

Affiliation: The Department of Chemistry and Oxford Centre for Integrative Systems Biology, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK.

ABSTRACT
2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components and in the hydroxylation of transcription factors and splicing factor proteins. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA and ribosomal proteins have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone N(ε)-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.

Show MeSH
Related in: MedlinePlus