The finding that oxygenase-catalyzed protein hydroxylation regulates animal transcription raises questions as to whether the translation machinery and prokaryotic proteins are analogously modified. Escherichia coli ycfD is a growth-regulating 2-oxoglutarate oxygenase catalyzing arginyl hydroxylation of the ribosomal protein Rpl16. Human ycfD homologs, Myc-induced nuclear antigen (MINA53) and NO66, are also linked to growth and catalyze histidyl hydroxylation of Rpl27a and Rpl8, respectively. This work reveals new therapeutic possibilities via oxygenase inhibition and by targeting modified over unmodified ribosomes.

2-Oxoglutarate (2OG)-dependent oxygenases play important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1,2, hydroxylation of transcription factors3, and of splicing factor proteins4. Recently, 2OG-oxygenases that catalyze hydroxylation of tRNA5-7 and ribosomal proteins8, have been shown to play roles in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9-12. The finding that the ribosomal oxygenases (ROX) occur in organisms ranging from prokaryotes to humans8 raises questions as to their structural and evolutionary relationships. In Escherichia coli, ycfD catalyzes arginine-hydroxylation in the ribosomal protein L16; in humans, Mina53 (MYC-induced nuclear antigen) and NO66 (Nucleolar protein 66) catalyze histidine-hydroxylation in ribosomal proteins rpL27a and rpL8, respectively. The functional assignments of the ROX open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in residue- and protein-selectivities of prokaryotic and eukaryotic ROX, crystal structures of ycfD and ycfDRM from E. coli and Rhodothermus marinus with those of human Mina53 and NO66 (hROX) reveal highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-oxygenases. ROX structures in complex with/without their substrates, support their functional assignments as hydroxylases, but not demethylases and reveal how the subfamily has evolved to catalyze the hydroxylation of different residue sidechains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-hydroxylases including the hypoxia-inducible factor asparaginyl-hydroxylase (FIH) and histone Nε-methyl lysine demethylases (KDMs) identifies branchpoints in 2OG-oxygenase evolution and distinguishes between JmjC-hydroxylases and -demethylases catalyzing 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 likely contributed to the evolution of the wide range of reactions catalyzed by iron-oxygenases.

The mechanisms by which gene expression is regulated by oxygen are of considerable interest from basic science and therapeutic perspectives. Using mass spectrometric analyses of Saccharomyces cerevisiae ribosomes, we found that the amino acid residue in closest proximity to the decoding center, Pro-64 of the 40S subunit ribosomal protein Rps23p (RPS23 Pro-62 in humans) undergoes posttranslational hydroxylation. We identify RPS23 hydroxylases as a highly conserved eukaryotic subfamily of Fe(II) and 2-oxoglutarate dependent oxygenases; their catalytic domain is closely related to transcription factor prolyl trans-4-hydroxylases that act as oxygen sensors in the hypoxic response in animals. The RPS23 hydroxylases in S. cerevisiae (Tpa1p), Schizosaccharomyces pombe and green algae catalyze an unprecedented dihydroxylation modification. This observation contrasts with higher eukaryotes, where RPS23 is monohydroxylated; the human Tpa1p homolog OGFOD1 catalyzes prolyl trans-3-hydroxylation. TPA1 deletion modulates termination efficiency up to ∼10-fold, including of pathophysiologically relevant sequences; we reveal Rps23p hydroxylation as its molecular basis. In contrast to most previously characterized accuracy modulators, including antibiotics and the prion state of the S. cerevisiae translation termination factor eRF3, Rps23p hydroxylation can either increase or decrease translational accuracy in a stop codon context-dependent manner. We identify conditions where Rps23p hydroxylation status determines viability as a consequence of nonsense codon suppression. The results reveal a direct link between oxygenase catalysis and the regulation of gene expression at the translational level. They will also aid in the development of small molecules altering translational accuracy for the treatment of genetic diseases linked to nonsense mutations. translation | hypoxia | ribosomal hydroxylation | 2-oxoglutarate oxygenase | nonsense readthrough T he rises in atmospheric oxygen levels provided life with a new energy source, but necessitated the evolution of regulatory mechanisms (1). Defining how cells regulate protein biosynthesis by the direct addition of oxygen to cellular molecules is of current basic and medicinal interest. Recent work suggests the presence of multiple regulatory levels and interfaces between O 2 and gene expression, many of which are catalyzed by Fe(II)-and 2-oxoglutarate (2OG)-dependent oxygenases, some of which are therapeutic targets (2). The hypoxic response in animals, but not lower organisms, is substantially mediated by prolyl trans-4-hydroxylation of the hypoxia-inducible transcription factor (HIF) (3-6). In addition to the gene-specific regulation of transcription via HIF, oxygen-dependent modifications at other interfaces have been discovered: hydroxylation of splicing-related proteins (7), histone lysyl demethylation (8), and 5-methylcytosine hydroxylation (9). In addition to signaling, posttranslational hydroxylations can also have structural roles: prolyl trans-4-hydroxylation stabilizes the c...

2-Oxoglutarate (2OG) and Fe(II)-dependent oxygenase domaincontaining protein 1 (OGFOD1) is predicted to be a conserved 2OG oxygenase, the catalytic domain of which is related to hypoxia-inducible factor prolyl hydroxylases. OGFOD1 homologs in yeast are implicated in diverse cellular functions ranging from oxygen-dependent regulation of sterol response genes (Ofd1, Schizosaccharomyces pombe) to translation termination/mRNA polyadenylation (Tpa1p, Saccharomyces cerevisiae). However, neither the biochemical activity of OGFOD1 nor the identity of its substrate has been defined. Here we show that OGFOD1 is a prolyl hydroxylase that catalyzes the posttranslational hydroxylation of a highly conserved residue (Pro-62) in the small ribosomal protein S23 (RPS23). Unusually OGFOD1 retained a high affinity for, and forms a stable complex with, the hydroxylated RPS23 substrate. Knockdown or inactivation of OGFOD1 caused a cell type-dependent induction of stress granules, translational arrest, and growth impairment in a manner complemented by wild-type but not inactive OGFOD1. The work identifies a human prolyl hydroxylase with a role in translational regulation.translational control | ribosome | 2-oxoglutarate oxygenase | hypoxia T he human genome encodes ∼60 2-oxoglutarate (2OG)-dependent oxygenases that catalyze diverse biological oxidations including hydroxylation of small molecules and proteins, and demethylation of histones and DNA/RNA (1). The identification of two types of 2OG oxygenase that regulate the transcriptional response to hypoxia by prolyl and asparaginyl hydroxylations in hypoxia-inducible factor (HIF), has led to the proposal that the hydroxylation of intracellular proteins may be involved in other signaling mechanisms (2). We have recently assigned 2OG oxygenases related to the HIF asparaginyl hydroxylase, factor-inhibiting HIF, as histidinyl and argininyl hydroxylases that catalyze hydroxylation of eukaryotic and prokaryotic ribosomes, respectively (3).2OG and Fe(II)-dependent oxygenase domain-containing protein 1 (OGFOD1) is a highly conserved 2OG oxygenase in eukaryotes. In the fission yeast, Schizosaccharomyces pombe, the homolog of OGFOD1, Ofd1, mediates oxygen-sensitive degradation of the N-terminal region of the transcription factor Sre1 [a homolog of sterol-response element-binding protein (SREBP)] after cleavage from the endoplasmic reticulum membrane, so contributing to oxygen-dependent regulation of the sterol response (4). The oxygen-sensitive SREBP pathway is not conserved in Saccharomyces cerevisiae (5), despite a conserved homolog of OGFOD1 [termination and polyadenylation 1 (Tpa1p)] in this species, suggesting Tpa1p has other roles. Indeed, TPA1 was identified in a screen for genes promoting stop codon readthrough (6). Tpa1p activity is linked to termination efficiency, mRNA polyadenylation, and mRNA stability. Structures of Tpa1p reveal the 2OG oxygenase characteristic doublestranded β-helix (DSBH) fold domain with typical Fe(II) and 2OG binding residues, but also indicate, as predicted f...

The FIH hydroxylase is a cellular peroxide sensor that modulates HIF transcriptional activityHIF asparaginyl hydroxylase (FIH) is shown to be strikingly more sensitive to peroxide than the HIF prolyl hydroxylases, indicating that hypoxia and oxidative stress are distinct regulators of the HIF response.