Ribosomal oxygenases are structurally conserved from prokaryotes to humans

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1,2 and in the hydroxylation of transcription factors3 and splicing factor proteins4. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA5,6,7 and ribosomal proteins8 have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9,10,11,12. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans8 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 Nε-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

Substance abusers' personality disorders and staff members' emotional reactions

<p>Abstract</p> <p>Background</p> <p>Previous research has indicated that aggressive behaviour and DSM-IV cluster B personality disorders (PD) may be associated with professionals' emotional reactions to clients, and that cluster C PD may be associated with positive emotional reactions.</p> <p>Methods</p> <p>Staff members recruited from workshops completed a self-report inventory of emotional reactions to patients, the Feeling Word Checklist-58, and substance abusers completed a self-report of DSM-IV personality disorder, the DSM-IV and ICD-10 Personality Disorder Questionnaire. Correlational analysis and multiple regression analysis was used to assess the associations between personality disorders and emotional reations.</p> <p>Results</p> <p>Cluster B disorder features were associated with feeling distance to patients, and cluster C disorder features were associated with feeling helpful towards patients. Cluster A disorders had no significant impact on emotional reactions.</p> <p>Conclusion</p> <p>The findings confirm clinical experiences that personality disorder features in patients with substance abuse have an impact on staff members reactions to them. These reactions should be considered in supervision of staff, and in treatment models for patients with co-morbid personality disorders and substance abuse.</p

Stringency of the 2-His–1-Asp Active-Site Motif in Prolyl 4-Hydroxylase

The non-heme iron(II) dioxygenase family of enzymes contain a common 2-His–1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C–H bonds. Prolyl 4-hydroxylase (P4H) is an α-ketoglutarate-dependent iron(II) dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His–1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase) and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change

Evidence for the slow reaction of hypoxia-inducible factor prolyl hydroxylase 2 with oxygen.

The response of animals to hypoxia is mediated by the hypoxia-inducible transcription factor. Human hypoxia-inducible factor is regulated by four Fe(II)- and 2-oxoglutarate-dependent oxygenases: prolyl hydroxylase domain enzymes 1-3 catalyse hydroxylation of two prolyl-residues in hypoxia-inducible factor, triggering its degradation by the proteasome. Factor inhibiting hypoxia-inducible factor catalyses the hydroxylation of an asparagine-residue in hypoxia-inducible factor, inhibiting its transcriptional activity. Collectively, the hypoxia-inducible factor hydroxylases negatively regulate hypoxia-inducible factor in response to increasing oxygen concentration. Prolyl hydroxylase domain 2 is the most important oxygen sensor in human cells; however, the underlying kinetic basis of the oxygen-sensing function of prolyl hydroxylase domain 2 is unclear. We report analyses of the reaction of prolyl hydroxylase domain 2 with oxygen. Chemical quench/MS experiments demonstrate that reaction of a complex of prolyl hydroxylase domain 2, Fe(II), 2-oxoglutarate and the C-terminal oxygen-dependent degradation domain of hypoxia-inducible factor-α with oxygen to form hydroxylated C-terminal oxygen-dependent degradation domain and succinate is much slower (approximately 100-fold) than for other similarly studied 2-oxoglutarate oxygenases. Stopped flow/UV-visible spectroscopy experiments demonstrate that the reaction produces a relatively stable species absorbing at 320 nm; Mössbauer spectroscopic experiments indicate that this species is likely not a Fe(IV)=O intermediate, as observed for other 2-oxoglutarate oxygenases. Overall, the results obtained suggest that, at least compared to other studied 2-oxoglutarate oxygenases, prolyl hydroxylase domain 2 reacts relatively slowly with oxygen, a property that may be associated with its function as an oxygen sensor