A human protein hydroxylase that accepts D -residues

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.