Structural basis for divergent C-H hydroxylation selectivity in two Rieske oxygenases

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Rieske oxygenases are enzymes capable of precise C-H hydroxylation, a critical reaction in biocatalysis.
• Understanding the structural basis of their substrate specificity and site-selectivity is vital for enzyme development.
• Limited structural data exists for Rieske oxygenases acting on diverse chemical scaffolds.

Purpose of the Study:

• To determine the structures of two Rieske monooxygenases, SxtT and GxtA, involved in paralytic shellfish toxin (PST) biosynthesis.
• To elucidate the structural features governing substrate positioning and reaction selectivity in these enzymes.
• To expand the structural knowledge of Rieske oxygenases, particularly those functionalizing complex natural product pathways.

Main Methods:

• X-ray crystallography was used to determine the structures of SxtT and GxtA.
• Substrate-bound structures were obtained to visualize enzyme-substrate interactions.
• Site-directed mutagenesis experiments were performed to probe the function of specific amino acid residues.

Main Results:

• The crystal structures of SxtT and GxtA were determined, adding to the limited structural repertoire of Rieske oxygenases.
• Substrate-bound structures revealed key interactions involved in positioning substrates within the enzyme's active site.
• Mutagenesis studies identified specific residues critical for the divergent catalytic selectivity observed between SxtT and GxtA.

Conclusions:

• The structures of SxtT and GxtA provide crucial insights into the mechanism of Rieske monooxygenases in PST biosynthesis.
• Specific amino acid residues play a significant role in controlling substrate binding and directing the regioselectivity of hydroxylation.
• This work enhances our understanding of enzyme selectivity and provides a foundation for engineering Rieske oxygenases for biocatalytic applications.