1. Observing extradiol dioxygenases in action through a crystalline lens.
- Author
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Campbell J and Wang Y
- Subjects
- Animals, Oxygenases metabolism, Oxygenases chemistry, Oxygenases genetics, Electron Spin Resonance Spectroscopy methods, Dioxygenases metabolism, Dioxygenases chemistry, Dioxygenases genetics, Lens, Crystalline enzymology, Lens, Crystalline metabolism
- Abstract
Extradiol dioxygenases are a class of non-heme iron-dependent enzymes found in eukaryotes and prokaryotes that play a vital role in the aerobic catabolism of aromatic compounds. They are generally divided into three evolutionarily independent superfamilies with different protein folds. Our recent studies have shed light on the catalytic mechanisms and structure-function relationships of two specific extradiol dioxygenases: 3-hydroxyanthranilate-3,4-dioxygenase, a Type III enzyme essential in mammals for producing a precursor for nicotinamide adenine dinucleotide, and L-3,4-dihydroxyphenylalanine dioxygenase, an uncommon form of Type I enzymes involved in natural product biosynthesis. This work details the expression and isolation methods for these extradiol dioxygenases and introduces approaches to achieve homogeneity and high occupancy of the enzyme metal centers. Techniques such as ultraviolet-visible and electron paramagnetic resonance spectroscopies, as well as oxygen electrode measurements, are discussed for probing the interaction of the non-heme iron center with ligands and characterizing enzymatic activities. Moreover, protein crystallization has been demonstrated as a powerful tool to study these enzymes. We highlight in crystallo reactions and single-crystal spectroscopic methods to further elucidate enzymatic functions and protein dynamics., (Copyright © 2024. Published by Elsevier Inc.)
- Published
- 2024
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