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Cofactor processing in galactose oxidase.

Susan Firbank1, Melanie Rogers, Ramon Hurtado Guerrero

  • 1Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.

Biochemical Society Symposium
|March 22, 2005
PubMed
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Galactose oxidase (GO) is a copper enzyme with a unique cofactor generated post-translationally. Its prosequence acts as a chaperone, guiding cofactor formation and copper binding for the mature enzyme.

Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Chemistry

Background:

  • Galactose oxidase (GO) is a 68 kDa monomeric enzyme from Fusarium graminearum, featuring a copper ion and an amino acid-derived cofactor.
  • Its catalytic mechanism as a radical enzyme has been extensively studied using structural, spectroscopic, kinetic, and mutational methods.
  • A key feature is the post-translational, autocatalytic generation of an organic cofactor from active-site amino acid residues, involving a thioether bond between Cys-228 and Tyr-272.

Purpose of the Study:

  • To investigate the role of the N-terminal prosequence in the biogenesis of the galactose oxidase cofactor.
  • To understand the structural and functional implications of the prosequence in copper binding and autocatalytic processing.
  • To elucidate the mechanism of mature GO formation from its pro-form.

Related Experiment Videos

Main Methods:

  • Purification of pro-galactose oxidase (pro GO) from a heterologous host (Aspergillus nidulans) under copper-free conditions.
  • Autocatalytic processing of pro GO in the presence of copper and oxygen to form mature GO.
  • Structural comparison of pro GO and mature GO using techniques like X-ray crystallography.
  • Analysis of active-site residue conformations and their changes during processing.

Main Results:

  • The prosequence is linked to the formation of the active-site thioether bond and cofactor generation.
  • Structural comparison revealed significant local differences in main-chain and side-chain positions between pro GO and mature GO, particularly in the active site.
  • The prosequence appears to function as an intramolecular chaperone, facilitating an open active-site structure for copper binding and cofactor chemistry.
  • Autocatalytic processing of GO can occur even without the prosequence, provided copper and oxygen are present.

Conclusions:

  • The N-terminal prosequence of galactose oxidase plays a crucial role as an intramolecular chaperone in the autocatalytic formation of its unique cofactor and copper binding.
  • Structural insights highlight the prosequence's influence on active-site conformation, essential for enzyme maturation.
  • The findings contribute to understanding radical enzyme biogenesis and the intricate mechanisms of post-translational modification.