A human protein hydroxylase that accepts D-residues

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Factor inhibiting hypoxia-inducible factor (FIH) is a key enzyme in regulating cellular responses to hypoxia.
• FIH functions as a 2-oxoglutarate-dependent protein hydroxylase, catalyzing C3 hydroxylations.

Purpose of the Study:

• To investigate the substrate specificity of FIH towards both L- and D-amino acid residues.
• To explore the structural and mechanistic basis for FIH's substrate selectivity.

Main Methods:

• Enzymatic assays using various L- and D-amino acid substrates.
• Mass spectrometry for product identification and quantification.
• X-ray crystallography to determine enzyme-substrate-product complexes.
• Density Functional Theory (DFT) calculations to model reaction mechanisms.

Main Results:

• FIH demonstrates promiscuity, accepting both L- and D-amino acid residues for hydroxylation.
• Distinct substrate selectivities were observed for D- and L-epimers, with examples including allylglycine and aspartate.
• Unexpected dihydroxylation of D-leucine was observed, contrasting with the monohydroxylation of L-leucine.
• Structural and computational studies provided insights into the molecular basis of this stereoselectivity.

Conclusions:

• The findings expand the known substrate scope of isolated FIH.
• This work implies the potential for engineering FIH variants with tailored substrate specificities.
• Understanding FIH's stereoselectivity is crucial for its role in hypoxia signaling and potential therapeutic applications.