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Updated: Oct 7, 2025
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Published on: October 3, 2018
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Design principles for site-selective hydroxylation by a Rieske oxygenase
Jianxin Liu1, Jiayi Tian1, Christopher Perry1,2,3
1Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA.
Nature Communications
|January 12, 2022
Summary
Rieske oxygenases SxtT and GxtA use distinct protein regions to control C-H hydroxylation site-selectivity. Key residues identified in SxtT can be altered to mimic GxtA
Area of Science:
- Biochemistry and enzymology
- Structural biology
- Organic chemistry
Background:
- Rieske oxygenases are iron-dependent enzymes catalyzing challenging C-H bond functionalization.
- Limited structural data exists for Rieske oxygenases, hindering understanding of their reaction diversity.
- Understanding enzyme structure-function relationships is crucial for biocatalyst development.
Purpose of the Study:
- To elucidate the structural basis for site-selectivity in Rieske oxygenases SxtT and GxtA.
- To identify specific protein regions and residues controlling C-H hydroxylation selectivity.
- To provide a framework for engineering Rieske oxygenases as biocatalysts.
Main Methods:
- High-resolution crystal structure determination of SxtT and GxtA with bound substrates.
- Xenon-pressurized crystallography to visualize substrate access pathways.
- Site-directed mutagenesis to investigate the role of identified residues in selectivity.
Main Results:
- Structural characterization of SxtT and GxtA revealed distinct active site architectures.
- A substrate access tunnel in GxtA was identified using Xenon-pressurized crystallography.
- Six key residues in three regions of SxtT were found to control C-H hydroxylation site-selectivity.
- A SxtT variant with mutations in these regions exhibited the non-native selectivity of GxtA.
Conclusions:
- Specific protein regions and residues dictate Rieske oxygenase site-selectivity.
- The identified selectivity determinants are conserved across related enzymes.
- This work enables predictive engineering and repurposing of Rieske oxygenases for biocatalysis.
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