BI87CH24_Schofield ARI 2 June 2018 14:41 Annual Review of Biochemistry 2-Oxoglutarate-Dependent Oxygenases Md. Saiful Islam,1,∗ Thomas M. Leissing,1,∗ Rasheduzzaman Chowdhury,1,∗ Richard J. Hopkinson,1,2,∗ and Christopher J. Schofield1,∗ 1The Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom; email: christopher.schofi[email protected] 2Current affiliation for Richard J. Hopkinson: Leicester Institute of Structural and Chemical Biology and Department of Chemistry, University of Leicester, Leicester LE1 7RH, United Kingdom; email: [email protected] Annu. Rev. Biochem. 2018. 87:585–620 Keywords First published as a Review in Advance on 2-oxoglutarate, α-ketoglutarate, oxygenase, dioxygenase, hydroxylation, March 1, 2018 demethylation, biosynthesis, oxygen sensing, hypoxia, epigenetics The Annual Review of Biochemistry is online at biochem.annualreviews.org Abstract https://doi.org/10.1146/annurev-biochem- 2-Oxoglutarate (2OG)-dependent oxygenases (2OGXs) catalyze a remark- 061516-044724 ably diverse range of oxidative reactions. In animals, these comprise hydrox- Copyright c 2018 by Annual Reviews. ylations and N-demethylations proceeding via hydroxylation; in plants and All rights reserved microbes, they catalyze a wider range including ring formations, rearrange- ∗ Access provided by West Virginia University on 02/26/19. For personal use only. Annu. Rev. Biochem. 2018.87:585-620. Downloaded from www.annualreviews.org All authors contributed equally to this review. ments, desaturations, and halogenations. The catalytic flexibility of 2OGXs is reflected in their biological functions. After pioneering work identified the roles of 2OGXs in collagen biosynthesis, research revealed they also ANNUAL function in plant and animal development, transcriptional regulation, nu- REVIEWS Further cleic acid modification/repair, fatty acid metabolism, and secondary metabo- Click here to view this article's online features: lite biosynthesis, including of medicinally important antibiotics. In plants, • Download figures as PPT slides • Navigate linked references 2OGXs are important agrochemical targets and catalyze herbicide degrada- • Download citations tion. Human 2OGXs, particularly those regulating transcription, are cur- • Explore related articles • Search keywords rent therapeutic targets for anemia and cancer. Here, we give an overview of the biochemistry of 2OGXs, providing examples linking to biological func- tion, and outline how knowledge of their enzymology is being exploited in medicine, agrochemistry, and biocatalysis. 585 BI87CH24_Schofield ARI 2 June 2018 14:41 Contents INTRODUCTION . 586 STRUCTURALANDMECHANISTICOVERVIEWOF2OGXs................. 586 OVERVIEWOF2OGXFUNCTIONS........................................... 592 2OG-DEPENDENT PROTEIN HYDROXYLASES INVOLVED INCOLLAGENBIOSYNTHESISANDRELATEDENZYMES.............. 593 HYPOXIA-INDUCIBLE FACTOR HYDROXYLASES ANDRELATEDENZYMES.................................................. 593 2OGXsASPROTEINDEMETHYLASES........................................ 598 2OGXsACTINGONNUCLEOBASESANDNUCLEICACIDS................. 601 2OGX-CATALYZEDSMALL-MOLECULEOXIDATIONS..................... 603 2OGX-CATALYZEDLIPIDOXIDATIONS..................................... 607 INHIBITION OF 2OGXs . 608 BIOCATALYTICAPPLICATIONSOF2OGXs.................................. 611 CONCLUSIONSANDFUTUREPROSPECTS.................................. 612 INTRODUCTION Following pioneering studies identifying their roles in collagen (1) and small-molecule biosynthe- sis, 2-oxoglutarate (2OG; also known as α-ketoglutarate)-dependent oxygenases (2OGXs) have emerged as a widely distributed superfamily in aerobic biology (2). Most (likely >95%) 2OGXs employ Fe(II) as a cofactor and 2OG and O2 as cosubstrates, producing CO2 and succinate as coproducts (Figure 1). In terms of both substrate and product selectivity, 2OGXs are among the most biochemically flexible enzymes (Figure 2) (3). 2OGXs catalyze oxidation of both oligomeric Supplemental Material substrates (proteins, nucleic acids, lipids) and small molecules, the latter in isolated form and when tethered to peptides (4). The most common 2OGX-catalyzed reactions are hydroxylations, including N-methyl,and,toalesserextent,O-methyl demethylations, which proceed via initial 2OG: 2-oxoglutarate hydroxylation (Figure 2; Supplemental Table 1) (5, 6). 2OGXs also catalyze more exotic trans- 2OGX: formations, commonly in secondary metabolism, where their roles are medicinally relevant, e.g., 2-oxoglutarate- during β-lactam biosynthesis. This review provides a concise overview of current knowledge of dependent structural, mechanistic, and functional studies on 2OGXs, guides the reader to specialist reviews, oxygenase and identifies avenues for future research. Oxygenase: enzyme Access provided by West Virginia University on 02/26/19. For personal use only. Annu. Rev. Biochem. 2018.87:585-620. Downloaded from www.annualreviews.org that catalyzes substrate oxidation with oxygen transfer from dioxygen STRUCTURAL AND MECHANISTIC OVERVIEW OF 2OGXs Double-stranded Extensive crystallographic analyses reveal 2OGX structures are characterized by a distorted β-helix (DSBH): double-stranded β-helix (DSBH; also known as jelly-roll) core fold (Figure 3) (6–10). The right- core fold of 2OGX; handed class I DSBH fold occurs in many protein classes and is the only one of four possi- also characteristic of ble DSBH folds observed in biology. The DSBH fold is also characteristic of cupin and JmjC JmjC and cupin-fold proteins proteins, many of which are 2OGXs (9). The DSBH fold in 2OGXs was predicted following studies on isopenicillin N synthase (IPNS), which does not use 2OG, but is structurally homol- ogous with and mechanistically related to 2OGXs. Crystallography of deacetoxycephalosporin C synthase (DAOCS) (11), taurine dioxygenase (12), and clavaminic acid synthase (13) confirmed the presence of the DSBH in 2OGXs and defined subfamily-characteristic features (Figure 3) (7–10). 586 Islam et al. BI87CH24_Schofield ARI 2 June 2018 14:41 O N H O O R O O FeIV His O O O His Asp/Glu O O CO 2 O N N N H H O• O H O CO O R 2 R O O HisO II His O O FeIII O Fe R His O O O O O FeIV His Asp/Glu His Asp/Glu O O His Asp/Glu O2 O O N N H OH R 2 HIF-α peptide O O II His O OH Fe R O O O O FeIII His O His Asp/Glu Pro-564 O His Asp/Glu H2O O Metal N 2OG O H N OH OH R 2 Asp-315 O O His O Arg-383 FeII R O O O O FeII His O His Asp/Glu O His-313 His-374 His Asp/Glu 2 H2O O O O O N OH2 2 H O H O O 2 R O HO OH 2 FeII His O FeII His OH O H2O O O His Asp/Glu His Asp/Glu O OH HO O Figure 1 Access provided by West Virginia University on 02/26/19. For personal use only. Annu. Rev. Biochem. 2018.87:585-620. Downloaded from www.annualreviews.org Consensus mechanism for 2OGX-catalyzed hydroxylation. Following bidentate coordination of 2OG to Fe(II), substrate binding (e.g., for a prolyl residue) proximal to Fe(II) normally weakens binding of a metal-ligated water molecule, providing a vacant coordination site for O2. Oxidative decarboxylation of 2OG forms succinate, CO2, and an Fe(IV)-oxo intermediate. The latter reacts with substrate C–H to form an alcohol concomitant with reduction of Fe(IV) to Fe(II). Note the coordination position of the 2OG C-1 carboxylate can vary (see text). (Inset) View from a structure of human PHD2 complexed with Mn(II) [substituting for Fe(II) in crystallography], 2OG, and a fragment of the HIF-α with the oxidized C–H bond shown projecting toward the metal (PDB ID: 5L9B). Abbreviations: 2OG, 2-oxoglutarate; 2OGX, 2OG-dependent oxygenase; HIF-α, hypoxia-inducible factor alpha; PDB, Protein Data Bank; PHD2, prolyl hydroxylase domain 2. The eight-stranded (I–VIII) 2OGX DSBH core fold has major (I, III, VI, VIII) and minor (II, IV, V, VII) β-sheets forming a squashed barrel, the more open end of which contains Fe(II) and 2OG binding elements that are normally isolated from solution (Figure 3) (7–10). The DSBH core is augmented by additional β-strands extending the major and, sometimes, minor β-sheets. α-Helices present at the N terminus of the DSBH play roles in fold stabilization and, sometimes, www.annualreviews.org • 2-Oxoglutarate-Dependent Oxygenases 587 BI87CH24_Schofield ARI 2 June 2018 14:41 Hydroxylation R R R = H H X H X H X H OH O X = N, O R = H R = H O R HO X H X HH + O Br/Cl HX Halogenation Demethylation Sequential oxidation HH HH O H H H H RR' RR' RR' Desaturation Epoxidation Ring formation RHN RHN R O R CO2H O O S O N S N O R' R' HO2CHO2C Ring expansion R HO O R N N CO2H CO2H Proteins O O Nucleic acids Small molecules Lipids Access provided by West Virginia University on 02/26/19. For personal use only. Annu. Rev. Biochem. 2018.87:585-620. Downloaded from www.annualreviews.org R OH Carbohydrates R O O O CO2H HO Figure 2 Overview of reactions catalyzed by 2-oxoglutarate-dependent oxygenases (2OGXs): hydroxylation, demethylation, sequential oxidation, ring formation, ring expansion, halogenation, epoxidation, and desaturation. Colored arrows represent reactions on protein (red ), nucleic acid (dark blue), small-molecule (orange), lipid ( green), or carbohydrate (light blue) substrates. Only hydroxylation and demethylation reactions are reported on protein and nucleic acid substrates (at least for natural reactions); lipids/carbohydrates undergo hydroxylation. Ring formation/expansion, halogenation, epoxidation, and desaturation reactions are reported (almost) only on small molecules. The scheme is not exhaustive, e.g., there is a 2OGX-catalyzed cyclic peroxidation. 588 Islam et al. BI87CH24_Schofield ARI 2 June 2018 14:41 a Fe(II) ligating Extended N- and residues β C-terminal regions II-βIII insert for some oxygenases Parallel HXD/E...H or antiparallel β-strands (can N participate in substrate binding) C βIV-βV insert I II VIIIVII III IV VI V K/R K R Involved in Subfamily characteristic substrate basic residue binds the binding 2OG C-5 carboxylate b βIV-βV insert Minor β-sheet (II,IV,V,VII) V H183 IV VII H124 II Active site Fe(II) D126 R192 VI Y141 III VIII N βII-βIII insert I Access provided by West Virginia University on 02/26/19. For personal use only.