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The 100s loop in factor-inhibiting-HIF-1 (FIH) enzyme, particularly Tyr102, is crucial for synchronizing oxygen activation and substrate hydroxylation, impacting hypoxia sensing. This finding offers insights into selective enzyme modulation for therapeutic strategies.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Factor-inhibiting-HIF-1 (FIH) enzyme modulates hypoxia sensing, a key process in diseases like cancer and ischemia.
  • FIH's function depends on tightly coupling O2 activation and succinate production with HIF-1α/CTAD substrate hydroxylation.

Purpose of the Study:

  • To investigate the role of the FIH 100s loop (residues 102-118) in maintaining the coupling between FIH's catalytic steps.
  • To elucidate the specific function of the hinge residue Tyr102 in FIH's hypoxia-sensing mechanism.

Main Methods:

  • Gaussian Network Model (iGNM) analysis to identify dynamic domains and hinge residues.
  • Enzymological and biophysical studies of FIH 100s loop mutants.
  • Site-directed mutagenesis (Y102F, Y102A) and kinetic analysis.

Main Results:

  • iGNM identified the 100s loop and Tyr102 as a potential substrate-positioning element.
  • Mutant Y102F showed kinetics similar to wild-type FIH, suggesting a steric role for Tyr102.
  • The Y102A mutant produced succinate and bound O2 normally but showed significantly reduced HIF-1α/CTAD substrate hydroxylation.

Conclusions:

  • FIH loop hinge residue Tyr102 is not essential for O2 activation.
  • The steric interaction of Tyr102 is critical for substrate positioning during productive hydroxylation.
  • Tyr102 plays a vital role in synchronizing O2 activation with substrate hydroxylation for FIH function.