Adventures in Defining Roles of Oxygenases in the Regulation of Protein Biosynthesis

Abstract

The 2-oxoglutarate (2OG) dependent oxygenases were first identified as having roles in the post-translational modification of procollagen in animals. Subsequently in plants and microbes, they were shown to have roles in the biosynthesis of many secondary metabolites, including signalling molecules and the penicillin/cephalosporin antibiotics. Crystallographic studies of microbial 2OG oxygenases and related enzymes, coupled to DNA sequence analyses, led to the prediction that 2OG oxygenases are widely distributed in aerobic biology. This personal account begins with examples of the roles of 2OG oxygenases in antibiotic biosynthesis, and then describes efforts to assign functions to other predicted 2OG oxygenases. In humans, 2OG oxygenases have been found to have roles in small molecule metabolism, as well as in the epigenetic regulation of protein and nucleic acid biosynthesis and function. The roles and functions of human 2OG oxygenases are compared, focussing on discussion of their substrate and product selectivities. The account aims to emphasize how scoping the substrate selectivity of, sometimes promiscuous, enzymes can provide insights into their functions and so enable therapeutic work.

Inhibition of the Oxygen-Sensing Asparaginyl Hydroxylase Factor Inhibiting Hypoxia-Inducible Factor: A Potential Hypoxia Response Modulating Strategy

“…20,21,48 Subsequently, the HIF-α CTAD binds in a manner positioning Asn803 adjacent to the Fe(II); the CTAD Asn803 is held by hydrogen bonds including with Asp201 and Gln239 of FIH. 20 It is proposed that O 2 binds to an open coordination site on Fe(II) (potentially induced by substrate binding as was precedented with other 2OG oxygenases 17,49 but notably not PHD2 41 ) yielding an Fe(III) linked superoxide. The distal superoxide oxygen then attacks the C-2 carbonyl of 2OG, resulting in oxidative decarboxylation with formation of succinate, carbon dioxide (which likely efficiently leaves the active site), and a Fe(IV)O intermediate, the latter of which is likely the active oxidant.…”

“…In the resting state, Fe­(II) is complexed by the 2-His-1-Asp triad (His199, His279, and Asp201) with the remaining three coordination sites occupied by 2–3 labile water molecules. , 2OG binds in a bidentate manner via its C1 carboxylate and C2 ketone oxygen forming an octahedral coordination structure. ,, Subsequently, the HIF-α CTAD binds in a manner positioning Asn803 adjacent to the Fe­(II); the CTAD Asn803 is held by hydrogen bonds including with Asp201 and Gln239 of FIH . It is proposed that O 2 binds to an open coordination site on Fe­(II) (potentially induced by substrate binding as was precedented with other 2OG oxygenases , but notably not PHD2) yielding an Fe­(III) linked superoxide. The distal superoxide oxygen then attacks the C-2 carbonyl of 2OG, resulting in oxidative decarboxylation with formation of succinate, carbon dioxide (which likely efficiently leaves the active site), and a Fe­(IV)O intermediate, the latter of which is likely the active oxidant. ,, The ferryl species then stereospecifically abstracts a hydrogen atom from C-3 of HIF-α Asn803 to form a substrate radical and ferric hydroxide; a radical rebound process then gives the hydroxylated product and restores the Fe­(II) state. , The hydroxylated product and succinate are then released, , and the FIH-Fe­(II) complex can transition into a new catalytic cycle (Figure ).…”

Role of prolyl hydroxylase domain proteins in bone metabolism

“…PHD homologs (PHD1/2/3), and factor inhibiting HIF [FIH]) are non-heme dioxygenases which sense oxygen via direct enzymatic interaction with dissolved oxygen [ [22] , [23] , [24] ]. PHD1/2/3 are Fe 2+ 2-oxoglutarate-dependent dioxygenases (2OGD) which represent the largest group of non-heme, Fe 2 + bound, oxidizing enzymes.…”

“…2OGD substrates are diverse and found in proteins, nucleic acids, lipids, carbohydrates, and small molecules. For example, cytoplasmic procollagen's vitamin C-dependent post-translational proline and lysine hydroxylation, modifications required for its tertiary folding and quaternary assembly is dependent on 2OGD processing [ 24 , 25 ], and nuclear epigenetic Jumonji C domain containing histone lysine demethylation (KDM) enzyme and 10–11 translocation hydroxylates (TET1-3) enzyme, vital for regional context-dependent promotion or suppression of transcription, are as well 2OGD enzymes [ 23 , 24 ].…”

“…2OGD regulates HIF transcription activity [ 7 , 23 , 24 , [29] , [30] , [31] ]. HIF is a heterodimer transcription cofactor composed of an 2OGD substrate targeted alpha-subunit isoform (HIF1α/826aa, HIF2α/870aa, HIF3α/667aa) and a constitutively expressed beta-subunit HIFβ.…”