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.

抑制缺氧诱导因子 (FIH) 的因子是一种 2-酮戊二酸依赖性蛋白羟化酶，可催化蛋白质残基的 C3 羟基化。我们报告 FIH 可以接受 ( D )- 和 ( L ) - 残基进行羟基化。FIH 的底物选择性对于 ( D ) 和 ( L ) 差向异构体不同，例如，( D )- 而不是 ( L )-烯丙基甘氨酸，相反地 ( L )- 但不是 ( D )-天冬氨酸，在测试中进行单羟基化序列上下文。( L )-Leu 含底物经历 FIH 催化的单羟基化，而 ( D)-Leu 出乎意料地经历了二羟基化。晶体学、质谱和 DFT 研究提供了对 FIH 对 ( L )- 和 ( D )- 残基的选择性的见解。这项工作的结果扩大了被分离的 FIH 羟基化的已知底物的潜在范围，并暗示有可能生成具有改变的选择性的 FIH 变体。