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Oxygen reactivity in flavoenzymes: context matters.

Claudia A McDonald1, Rebecca L Fagan, François Collard

  • 1Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.

Journal of the American Chemical Society
|October 1, 2011
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Summary

Enzymes containing flavin cofactors react rapidly with oxygen. A positive charge near the flavin

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Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Published on: November 8, 2019

Area of Science:

  • Biochemistry
  • Enzymology
  • Bioinorganic Chemistry

Background:

  • Flavoenzymes, including oxidases and monooxygenases, exhibit faster reactions with oxygen compared to free flavins.
  • The role of a positive charge near the N5 position of the reduced flavin has been proposed as a key factor in this enhanced reactivity.
  • Understanding these mechanisms is crucial for elucidating enzyme function and evolution.

Purpose of the Study:

  • To investigate the role of a positively charged lysine residue near the flavin N5 in oxygen activation by flavoenzymes.
  • To compare the oxygen reactivity of wild-type fructosamine oxidase and dihydroorotate dehydrogenases with their respective lysine mutants.
  • To determine if a positive charge alone is sufficient for enhanced oxygen reactivity or if specific enzyme context is required.

Main Methods:

  • Site-directed mutagenesis was used to create lysine-to-methionine mutants (Lys276Met) in fructosamine oxidase.
  • Kinetic assays were performed to measure the rate constants for the reaction of wild-type and mutant enzymes with oxygen.
  • Comparative analysis of oxygen reaction rates was conducted for fructosamine oxidase and dihydroorotate dehydrogenases from E. coli and L. lactis.

Main Results:

  • Fructosamine oxidase exhibited a high rate constant for oxygen reaction (1.6 × 10^5 M^-1 s^-1), which was drastically reduced in the Lys276Met mutant (291 M^-1 s^-1), indicating the lysine's importance.
  • Dihydroorotate dehydrogenases from E. coli and L. lactis also showed significant oxygen reaction rates (6.2 × 10^4 and 3.0 × 10^3 M^-1 s^-1, respectively).
  • However, their corresponding lysine mutants (Lys66Met and Lys43Met) displayed rate constants similar to the wild-type enzymes, suggesting context-dependent function.

Conclusions:

  • The presence of a lysine residue near the flavin N5 is not sufficient on its own to guarantee enhanced oxygen reactivity.
  • The specific enzyme environment or "context" plays a critical role in how this lysine facilitates oxygen activation.
  • Evolution has employed diverse strategies to achieve the rapid reaction of flavins with oxygen in different enzymes.