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Christopher J. Schofield - One of the best experts on this subject based on the ideXlab platform.
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structure function relationships of human jmjc Oxygenases demethylases versus hydroxylases
Current Opinion in Structural Biology, 2016Co-Authors: Suzana Markolovic, Thomas M Leissing, Rasheduzzaman Chowdhury, Sarah E Wilkins, Christopher J. SchofieldAbstract:The Jumonji-C (JmjC) subfamily of 2-oxoglutarate (2OG)-dependent Oxygenases are of biomedical interest because of their roles in the regulation of gene expression and protein biosynthesis. Human JmjC 2OG Oxygenases catalyze oxidative modifications to give either chemically stable alcohol products, or in the case of Nɛ-methyl lysine demethylation, relatively unstable hemiaminals that fragment to give formaldehyde and the demethylated product. Recent work has yielded conflicting reports as to whether some JmjC Oxygenases catalyze N-methyl group demethylation or hydroxylation reactions. We review JmjC oxygenase-catalyzed reactions within the context of structural knowledge, highlighting key differences between hydroxylases and demethylases, which have the potential to inform on the possible type(s) of reactions catalyzed by partially characterized or un-characterized JmjC Oxygenases in humans and other organisms.
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studies on deacetoxycephalosporin c synthase support a consensus mechanism for 2 oxoglutarate dependent Oxygenases
Biochemistry, 2014Co-Authors: Hanna Tarhonskaya, Christopher J. Schofield, Rasheduzzaman Chowdhury, Timothy D W Claridge, Ivanhoe K. H. Leung, Luc Henry, Andrea Szollossi, Jacob T Bush, Aman Iqbal, Emily FlashmanAbstract:Deacetoxycephalosporin C synthase (DAOCS) catalyzes the oxidative ring expansion of penicillin N (penN) to give deacetoxycephalosporin C (DAOC), which is the committed step in the biosynthesis of the clinically important cephalosporin antibiotics. DAOCS belongs to the family of non-heme iron(II) and 2-oxoglutarate (2OG) dependent Oxygenases, which have substantially conserved active sites and are proposed to employ a consensus mechanism proceeding via formation of an enzyme·Fe(II)·2OG·substrate ternary complex. Previously reported kinetic and crystallographic studies led to the proposal of an unusual “ping-pong” mechanism for DAOCS, which was significantly different from other members of the 2OG oxygenase superfamily. Here we report pre-steady-state kinetics and binding studies employing mass spectrometry and NMR on the DAOCS-catalyzed penN ring expansion that demonstrate the viability of ternary complex formation in DAOCS catalysis, arguing for the generality of the proposed consensus mechanism for 2OG o...
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Inhibition of 2-oxoglutarate dependent Oxygenases
Chemical Society reviews, 2011Co-Authors: Nathan R. Rose, Michael A. Mcdonough, Oliver N. King, Akane Kawamura, Christopher J. SchofieldAbstract:2-Oxoglutarate (2OG) dependent Oxygenases are ubiquitous iron enzymes that couple substrate oxidation to the conversion of 2OG to succinate and carbon dioxide. In humans their roles include collagen biosynthesis, fatty acid metabolism, DNA repair, RNA and chromatin modifications, and hypoxic sensing. Commercial applications of 2OG oxygenase inhibitors began with plant growth retardants, and now extend to a clinically used pharmaceutical compound for cardioprotection. Several 2OG Oxygenases are now being targeted for therapeutic intervention for diseases including anaemia, inflammation and cancer. In this critical review, we describe studies on the inhibition of 2OG Oxygenases, focusing on small molecules, and discuss the potential of 2OG Oxygenases as therapeutic targets (295 references).
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Structural and Mechanistic Studies on γ-Butyrobetaine Hydroxylase
Chemistry & Biology, 2010Co-Authors: Ivanhoe K. H. Leung, Michael A. Mcdonough, Timothy D W Claridge, Tobias Krojer, Grazyna Kochan, Luc Henry, Frank Von Delft, Udo Oppermann, Christopher J. SchofieldAbstract:The final step in carnitine biosynthesis is catalyzed by γ-butyrobetaine (γBB) hydroxylase (BBOX), an iron/2-oxoglutarate (2OG) dependent oxygenase. BBOX is inhibited by trimethylhydrazine-propionate (THP), a clinically used compound. We report structural and mechanistic studies on BBOX and its reaction with THP. Crystallographic and sequence analyses reveal that BBOX and trimethyllysine hydroxylase form a subfamily of 2OG Oxygenases that dimerize using an N-terminal domain. The crystal structure reveals the active site is enclosed and how THP competes with γBB. THP is a substrate giving formaldehyde (supporting structural links with histone demethylases), dimethylamine, malonic acid semi-aldehyde, and an unexpected product with an additional carbon-carbon bond resulting from N-demethylation coupled to oxidative rearrangement, likely via an unusual radical mechanism. The results provide a basis for development of improved BBOX inhibitors and may inspire the discovery of additional rearrangement reactions.
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2 oxoglutarate Oxygenases are inhibited by a range of transition metals
Metallomics, 2010Co-Authors: Rok Sekirnik, Nathan R. Rose, Jasmin Mecinovic, Christopher J. SchofieldAbstract:2-Oxoglutarate Oxygenases are inhibited by a range of transition metals, as exemplified by studies on human histone demethylases and prolyl hydroxylase domain 2 (PHD2 or EGLN1). The biological effects associated with 2-oxoglutarate oxygenase inhibition may result from inhibition of more than one enzyme and by mechanisms in addition to simple competition with the Fe(II) cofactor.
Donald P Weeks - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of dicamba monooxygenase a rieske nonheme oxygenase that catalyzes oxidative demethylation
Journal of Molecular Biology, 2009Co-Authors: Razvan Dumitru, Wen Zhi Jiang, Donald P Weeks, Mark A WilsonAbstract:Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a widely used herbicide that is efficiently degraded by soil microbes. These microbes use a novel Rieske nonheme oxygenase, dicamba monooxygenase (DMO), to catalyze the oxidative demethylation of dicamba to 3,6-dichlorosalicylic acid (DCSA) and formaldehyde. We have determined the crystal structures of DMO in the free state, bound to its substrate dicamba, and bound to the product DCSA at 2.10-1.75 A resolution. The structures show that the DMO active site uses a combination of extensive hydrogen bonding and steric interactions to correctly orient chlorinated, ortho-substituted benzoic-acid-like substrates for catalysis. Unlike other Rieske aromatic Oxygenases, DMO oxygenates the exocyclic methyl group, rather than the aromatic ring, of its substrate. This first crystal structure of a Rieske demethylase shows that the Rieske oxygenase structural scaffold can be co-opted to perform varied types of reactions on xenobiotic substrates.
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a three component dicamba o demethylase from pseudomonas maltophilia strain di 6 gene isolation characterization and heterologous expression
Journal of Biological Chemistry, 2005Co-Authors: Patricia L Herman, Sarbani Chakraborty, Mark Behrens, Brenda M Chrastil, Joseph J Barycki, Donald P WeeksAbstract:Abstract Dicamba O-demethylase is a multicomponent enzyme from Pseudomonas maltophilia, strain DI-6, that catalyzes the conversion of the widely used herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid) to DCSA (3,6-dichlorosalicylic acid). We recently described the biochemical characteristics of the three components of this enzyme (i.e. reductaseDIC, ferredoxinDIC, and oxygenaseDIC) and classified the oxygenase component of dicamba O-demethylase as a member of the Rieske non-heme iron family of Oxygenases. In the current study, we used N-terminal and internal amino acid sequence information from the purified proteins to clone the genes that encode dicamba O-demethylase. Two reductase genes (ddmA1 and ddmA2) with predicted amino acid sequences of 408 and 409 residues were identified. The open reading frames encode 43.7- and 43.9-kDa proteins that are 99.3% identical to each other and homologous to members of the FAD-dependent pyridine nucleotide reductase family. The ferredoxin coding sequence (ddmB) specifies an 11.4-kDa protein composed of 105 residues with similarity to the adrenodoxin family of [2Fe-2S] bacterial ferredoxins. The oxygenase gene (ddmC) encodes a 37.3-kDa protein composed of 339 amino acids that is homologous to members of the Phthalate family of Rieske non-heme iron Oxygenases that function as monoOxygenases. Southern analysis localized the oxygenase gene to a megaplasmid in cells of P. maltophilia. Mixtures of the three highly purified recombinant dicamba O-demethylase components overexpressed in Escherichia coli converted dicamba to DCSA with an efficiency similar to that of the native enzyme, suggesting that all of the components required for optimal enzymatic activity have been identified. Computer modeling suggests that oxygenaseDIC has strong similarities with the core αsubunits of naphthalene 1,2-dioxygenase. Nonetheless, the present studies point to dicamba O-demethylase as an enzyme system with its own unique combination of characteristics.
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a three component dicamba o demethylase from pseudomonas maltophilia strain di 6 purification and characterization
Archives of Biochemistry and Biophysics, 2005Co-Authors: Sarbani Chakraborty, Mark Behrens, Patricia L Herman, A F Arendsen, Wilfred R Hagen, Deborah L Carlson, Xiao Zhuo Wang, Donald P WeeksAbstract:Abstract Dicamba O -demethylase is a multicomponent enzyme that catalyzes the conversion of the herbicide 2-methoxy-3,6-dichlorobenzoic acid (dicamba) to 3,6-dichlorosalicylic acid (DCSA). The three components of the enzyme were purified and characterized. Oxygenase DIC is a homotrimer (α) 3 with a subunit molecular mass of approximately 40 kDa. Ferredoxin DIC and reductase DIC are monomers with molecular weights of approximately 14 and 45 kDa, respectively. EPR spectroscopic analysis suggested the presence of a single [2Fe–2S] (2+/1+) cluster in ferredoxin DIC and a single Rieske [2Fe–2S] (2+; 1+) cluster within oxygenase DIC . Consistent with the presence of a Rieske iron–sulfur cluster, oxygenase DIC displayed a high reduction potential of E m,7.0 = −21 mV whereas ferredoxin DIC exhibited a reduction potential of approximately E m,7.0 = −171 mV. Optimal oxygenase DIC activity in vitro depended on the addition of Fe 2+ . The identification of formaldehyde and DCSA as reaction products demonstrated that dicamba O -demethylase acts as a monooxygenase. Taken together, these data suggest that oxygenase DIC is an important new member of the Rieske non-heme iron family of Oxygenases.
Michel Sylvestre - One of the best experts on this subject based on the ideXlab platform.
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Structural insight into the expanded PCB-degrading abilities of a biphenyl dioxygenase obtained by directed evolution.
Journal of Molecular Biology, 2011Co-Authors: Pravindra Kumar, Mahmood Mohammadi, Jean-françois Viger, Diane Barriault, Leticia Gomez-gil, Lindsay D Eltis, Jeffrey T Bolin, Michel SylvestreAbstract:The biphenyl dioxygenase of Burkholderia xenovorans LB400 is a multicomponent Rieske-type oxygenase that catalyzes the dihydroxylation of biphenyl and many polychlorinated biphenyls (PCBs). The structural bases for the substrate specificity of the enzyme's oxygenase component (BphAE(LB400)) are largely unknown. BphAE(p4), a variant previously obtained through directed evolution, transforms several chlorobiphenyls, including 2,6-dichlorobiphenyl, more efficiently than BphAE(LB400), yet differs from the parent oxygenase at only two positions: T335A/F336M. Here, we compare the structures of BphAE(LB400) and BphAE(p4) and examine the biochemical properties of two BphAE(LB400) variants with single substitutions, T335A or F336M. Our data show that residue 336 contacts the biphenyl and influences the regiospecificity of the reaction, but does not enhance the enzyme's reactivity toward 2,6-dichlorobiphenyl. By contrast, residue 335 does not contact biphenyl but contributes significantly to expansion of the enzyme's substrate range. Crystal structures indicate that Thr335 imposes constraints through hydrogen bonds and nonbonded contacts to the segment from Val320 to Gln322. These contacts are lost when Thr is replaced by Ala, relieving intramolecular constraints and allowing for significant movement of this segment during binding of 2,6-dichlorobiphenyl, which increases the space available to accommodate the doubly ortho-chlorinated congener 2,6-dichlorobiphenyl. This study provides important insight about how Rieske-type Oxygenases can expand substrate range through mutations that increase the plasticity and/or mobility of protein segments lining the catalytic cavity.
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factors affecting the enhancement of pcb degradative ability of soil microbial populations
International Biodeterioration & Biodegradation, 1994Co-Authors: Benoit Guilbeault, Mohammad Sondossi, Darakhshan Ahmad, Michel SylvestreAbstract:Abstract In this study, we compared the capacity of freshly isolated microbial populations obtained from soils with a history of contamination and evaluated the effect of analog enrichment on the potential of PCB degradation attained by the enhanced populations. It has previously been suggested that PCB degrading ability should improve through mutations of the oxygenase that can increase reactivity of the enzyme towards various individual congeners. Our results show that the chlorine content of the PCB mixture and duration of soil contamination (number of years) have no effect on the natural evolution of the ability of soil microbial populations to degrade higher chlorinated PCBs. Moreover, although our results provided evidence that analog enrichment can favor the selection of Oxygenases with increased activities, we failed to select for populations that are able to degrade higher chlorinated PCBs more efficiently. Our data also clearly showed that chlorobenzoates, the end-products of the chlorobiphenyl degradation pathway (or their metabolites) interfere with PCB degradation. Therefore, the efficiency of PCB degradation is not only impaired by the substrate selectivity pattern of the chlorobiphenyl Oxygenases, but also by the stringent control of metabolite production and it is likely that population adjustment to prevent production of metabolites that could interfere with PCB degradation would be difficult to achieve through natural selection.
Martin Münzel - One of the best experts on this subject based on the ideXlab platform.
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Aspartate/asparagine-β-hydroxylase crystal structures reveal an unexpected epidermal growth factor-like domain substrate disulfide pattern
Nature Communications, 2019Co-Authors: Inga Pfeffer, Ks Hewitson, Lennart Brewitz, Tobias Krojer, Sacha A. Jensen, Grazyna T. Kochan, Nadia J. Kershaw, Luke A. Mcneill, Holger Kramer, Martin MünzelAbstract:AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1–3, 2–4, 5–6 disulfide bonding pattern; an unexpected Cys3–4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG Oxygenases. AspH catalyses hydroxylation of asparagine and aspartate residues in epidermal growth factor-like domains (EGFDs). Here, the authors present crystal structures of AspH with and without substrates and show that AspH uses EFGD substrates with a non-canonical disulfide pattern.
Tobias Krojer - One of the best experts on this subject based on the ideXlab platform.
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Aspartate/asparagine-β-hydroxylase crystal structures reveal an unexpected epidermal growth factor-like domain substrate disulfide pattern
Nature Communications, 2019Co-Authors: Inga Pfeffer, Ks Hewitson, Lennart Brewitz, Tobias Krojer, Sacha A. Jensen, Grazyna T. Kochan, Nadia J. Kershaw, Luke A. Mcneill, Holger Kramer, Martin MünzelAbstract:AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1–3, 2–4, 5–6 disulfide bonding pattern; an unexpected Cys3–4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG Oxygenases. AspH catalyses hydroxylation of asparagine and aspartate residues in epidermal growth factor-like domains (EGFDs). Here, the authors present crystal structures of AspH with and without substrates and show that AspH uses EFGD substrates with a non-canonical disulfide pattern.
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Ribosomal Oxygenases are structurally conserved from prokaryotes to humans
Nature, 2014Co-Authors: Rasheduzzaman Chowdhury, Rok Sekirnik, Ian J Clifton, Tobias Krojer, Nadia J. Kershaw, Nigel C Brissett, Chia-hua Ho, Stanley S. Ng, Wei Ge, J R C MunizAbstract:Crystal structures of human and prokaryotic ribosomal Oxygenases reported here, with and without their ribosomal protein substrates, support their assignments as hydroxylases, and provide insights into the evolution of the JmjC-domain-containing hydroxylases and demethylases. 2-Oxoglutarate (2OG)-dependent Oxygenases have important roles in the regulation of gene expression via demethylation of N -methylated chromatin components^ 1 , 2 and in the hydroxylation of transcription factors^ 3 and splicing factor proteins^ 4 . Recently, 2OG-dependent Oxygenases that catalyse hydroxylation of transfer RNA^ 5 , 6 , 7 and ribosomal proteins^ 8 have been shown to be important in translation relating to cellular growth, T_H17-cell differentiation and translational accuracy^ 9 , 10 , 11 , 12 . The finding that ribosomal Oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans^ 8 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. Christopher Schofield and colleagues present a comprehensive structural study of a recently discovered family of 2-oxoglutarate and iron-dependent Oxygenases termed ribosomal Oxygenases (ROXs). They have solved 13 new structures of human and bacterial ROXs with or without substrate. While the analysis confirms their assignment as hydroxylases, comparison with JmjC-domain-containing hydroxylases and demethylases provides an understanding of the evolution of these related families of proteins, in part driven by flexibility in the coordination position. The general binding mode of the hydroxylated residues is conserved between prokaryotic and human ROXs.
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Structural and Mechanistic Studies on γ-Butyrobetaine Hydroxylase
Chemistry & Biology, 2010Co-Authors: Ivanhoe K. H. Leung, Michael A. Mcdonough, Timothy D W Claridge, Tobias Krojer, Grazyna Kochan, Luc Henry, Frank Von Delft, Udo Oppermann, Christopher J. SchofieldAbstract:The final step in carnitine biosynthesis is catalyzed by γ-butyrobetaine (γBB) hydroxylase (BBOX), an iron/2-oxoglutarate (2OG) dependent oxygenase. BBOX is inhibited by trimethylhydrazine-propionate (THP), a clinically used compound. We report structural and mechanistic studies on BBOX and its reaction with THP. Crystallographic and sequence analyses reveal that BBOX and trimethyllysine hydroxylase form a subfamily of 2OG Oxygenases that dimerize using an N-terminal domain. The crystal structure reveals the active site is enclosed and how THP competes with γBB. THP is a substrate giving formaldehyde (supporting structural links with histone demethylases), dimethylamine, malonic acid semi-aldehyde, and an unexpected product with an additional carbon-carbon bond resulting from N-demethylation coupled to oxidative rearrangement, likely via an unusual radical mechanism. The results provide a basis for development of improved BBOX inhibitors and may inspire the discovery of additional rearrangement reactions.
