Fluorinated derivatives of pyridine-2,4-dicarboxylate are potent inhibitors of human 2-oxoglutarate dependent oxygenases.

2-Oxoglutarate (2OG) oxygenases have important roles in human biology and are validated medicinal chemistry targets. Improving the selectivity profile of broad-spectrum 2OG oxygenase inhibitors may help enable the identification of selective inhibitors for use in functional assignment work. We report the synthesis of F- and CF3-substituted derivatives of the broad-spectrum 2OG oxygenase inhibitor pyridine-2,4-dicarboxylate (2,4-PDCA). Their inhibition selectivity profile against selected functionally distinct human 2OG oxygenases was determined using mass spectrometry-based assays. F-substituted 2,4-PDCA derivatives efficiently inhibit the 2OG oxygenases aspartate/asparagine-ß-hydroxylase (AspH) and the JmjC lysine-specific N ε-demethylase 4E (KDM4E); The F- and CF3-substituted 2,4-PDCA derivatives were all less efficient inhibitors of the tested 2OG oxygenases than 2,4-PDCA itself, except for the C5 F-substituted 2,4-PDCA derivative which inhibited AspH with a similar efficiency as 2,4-PDCA. Notably, the introduction of a F- or CF3-substituent at the C5 position of 2,4-PDCA results in a substantial increase in selectivity for AspH over KDM4E compared to 2,4-PDCA. Crystallographic studies inform on the structural basis of our observations, which exemplifies how a small change on a 2OG analogue can make a substantial difference in the potency of 2OG oxygenase inhibition.

Growing with the wind. Ribosomal protein hydroxylation and cell growth.

In this Extra View we comment on our recent work on Sudestada1 (Sud1), a Drosophila 2-oxoglutarate (2OG)-dependent dioxygenase that belongs to the Ribosomal Oxygenase (ROX) subfamily. Sud1 is required for normal growth in Drosophila, and is conserved in yeast and mammals. We reported that Sud1 hydroxylates the ribosomal protein S23 (RPS23), and that its loss of function restricts growth and provokes activation of the unfolded protein response, apoptosis and autophagy. In this Extra View we speculate on the role that RPS23 hydroxylation might play in stop codon recognition and on the possible link between Sud1 loss-of-function and activation of the Unfolded Protein Response, Stress Granules formation and growth impairment.

Structure and functional analysis of YcfD, a novel 2-oxoglutarate/Fe²âº-dependent oxygenase involved in translational regulation in Escherichia coli.

The 2-oxoglutarate (2OG)/Fe²âº-dependent oxygenases (2OG oxygenases) are a large family of proteins that share a similar overall three-dimensional structure and catalyze a diverse array of oxidation reactions. The Jumonji C (JmjC)-domain-containing proteins represent an important subclass of the 2OG oxygenase family that typically catalyze protein hydroxylation; however, recently, other reactions have been identified, such as tRNA modification. The Escherichia coli gene, ycfD, was predicted to be a JmjC-domain-containing protein of unknown function based on primary sequence. Recently, YcfD was determined to act as a ribosomal oxygenase, hydroxylating an arginine residue on the 50S ribosomal protein L-16 (RL-16). We have determined the crystal structure of YcfD at 2.7 Å resolution, revealing that YcfD is structurally similar to known JmjC proteins and possesses the characteristic double-stranded ß-helix fold or cupin domain. Separate from the cupin domain, an additional globular module termed α-helical arm mediates dimerization of YcfD. We further have shown that 2OG binds to YcfD using isothermal titration calorimetry and identified key binding residues using mutagenesis that, together with the iron location and structural similarity with other cupin family members, allowed identification of the active site. Structural homology to ribosomal assembly proteins combined with GST (glutathione S-transferase)-YcfD pull-down of a ribosomal protein and docking of RL-16 to the YcfD active site support the role of YcfD in regulation of bacterial ribosome assembly. Furthermore, overexpression of YcfD is shown to inhibit cell growth signifying a toxic effect on ribosome assembly.

Post-translational hydroxylation by 2OG/Fe(II)-dependent oxygenases as a novel regulatory mechanism in bacteria.

Protein hydroxylation has been well-studied in eukaryotic systems. The structural importance of hydroxylation of specific proline and lysine residues during collagen biosynthesis is well established. Recently, key roles for post-translational hydroxylation in signaling and degradation pathways have been discovered. The function of hydroxylation in signaling is highlighted by its role in the hypoxic response of eukaryotic cells, where oxygen dependent hydroxylation of the hypoxia inducible transcription factor both targets it for degradation and blocks its activation. In contrast, the role of protein hydroxylation has been largely understudied in prokaryotes. Recently, an evolutionarily conserved class of ribosomal oxygenases (ROX) that catalyze the hydroxylation of specific residues in the ribosome has been identified in bacteria. ROX activity has been linked to cell growth, and has been found to have a direct impact on bulk protein translation. This discovery of ribosomal protein hydroxylation in bacteria could lead to new therapeutic targets for regulating bacterial growth, as well as, shed light on new prokaryotic hydroxylation signaling pathways. In this review, recent structural and functional studies will be highlighted and discussed, underscoring the regulatory potential of post-translational hydroxylation in bacteria.