A human protein hydroxylase that accepts D-residues

Abstract

Factor inhibiting hypoxia-inducible factor (FIH) is a 2-oxoglutarate-dependent protein hydroxylase that catalyses C3 hydroxylations of protein residues. We report FIH can accept (D)- and (L)-residues for hydroxylation. The substrate selectivity of FIH differs for (D) and (L) epimers, e.g., (D)- but not (L)-allylglycine, and conversely (L)- but not (D)-aspartate, undergo monohydroxylation, in the tested sequence context. The (L)-Leu-containing substrate undergoes FIH-catalysed monohydroxylation, whereas (D)-Leu unexpectedly undergoes dihydroxylation. Crystallographic, mass spectrometric, and DFT studies provide insights into the selectivity of FIH towards (L)- and (D)-residues. The results of this work expand the potential range of known substrates hydroxylated by isolated FIH and imply that it will be possible to generate FIH variants with altered selectivities.

2-Oxoglutarate derivatives can selectively enhance or inhibit the activity of human oxygenases

“…Movement of the Tyr102 FIH sidechain has been previously observed in crystal structures of FIH complexed with different substrates (Supplementary Fig. 33) 43 . In agreement with the proposal that the coordination of ( S )- 11 and ( S )- 18 to the FIH active site impairs substrate binding, FIH crystals with both inhibitor and substrate-bound could not be obtained.…”

“…The current evidence suggests that FIH is biochemically more promiscuous with respect to its substrate requirements than AspH as it also catalyzes the hydroxylation of residues other than Asp and Asn (Fig. 1b ) 41 – 43 .…”

Factor inhibiting HIF can catalyze two asparaginyl hydroxylations in VNVN motifs of ankyrin fold proteins

“…Interestingly, the yeast homolog of human OGFOD1 is reported to catalyze both the single and apparent double hydroxylation of the analogous prolyl residue ( 30 ). In the case of substrate analog studies with isolated FIH, we have also observed that the double hydroxylation of a single residue can occur ( d -Leu) though there is no evidence that this reaction is of biological relevance ( 27 ). Evidence that FIH can catalyze the desaturation of certain residues has also been accrued ( 17 ).…”

“…However, certain 2OG-dependent protein hydroxylases, including FIH, AspH, JMJD6 and some procollagen-modifying enzymes, likely act on multiple substrates ( 24 , 25 ). FIH is of particular interest because not only can it accept both HIF-α isoforms and multiple ARD (and other) proteins but also it can accept different residues as substrates, including Asn, Asp, His, and, at least in isolated form, some D-amino acids ( 17 , 18 , 26 , 27 ) at least in the context of ARD-derived substrates.…”

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

“…These broad-spectrum inhibitors show distinct selectivity profiles; for example, 2,4-PDCA efficiently inhibits AspH and some JmjC KDMs [ 14 , 15 ]. By contrast, 2,4-PDCA is only a weak inhibitor of the PHDs and of FIH [15] , which catalyzes the stereoselective C3 hydroxylation of Asn/Asp/His/Ser/Leu-residues (Supporting Figure S1) [ 16 , 17 ]. For other classes of human 2OG oxygenases, such as the ribosomal oxygenase 2 (RIOX2; MYC-induced nuclear antigen 53, MINA53), which catalyzes the stereoselective C3 hydroxylation of His39 of the 60S ribosomal protein L27a (RPL27A) [18] , the potency of these broad-spectrum inhibitors has not been reported.…”