Characterization of a second class Ie ribonucleotide reductase

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Class I ribonucleotide reductases (RNRs) are essential enzymes converting ribonucleotides to deoxyribonucleotides.
• Most RNRs utilize a metal cofactor in the R2 subunit for radical generation.
• A metal-free subclass, RNR Ie, features substitutions in conserved metal-binding residues.

Purpose of the Study:

• To biochemically and structurally characterize the R2e_QSK variant of metal-free Class I RNR.
• To investigate the organismal distribution and functional significance of R2e variants.
• To elucidate the mechanism of radical generation in R2e_QSK, particularly in Gardnerella vaginalis.

Main Methods:

• Biochemical assays to assess RNR activity.
• Site-directed mutagenesis and protein expression.
• Analysis of protein modification (tyrosine to DOPA).
• X-ray crystallography to determine protein structures.

Main Results:

• Identified and characterized the R2e_QSK variant, distinct from the previously studied VPK variant.
• Found R2e_QSK is crucial for deoxyribonucleotide synthesis in numerous organisms.
• Demonstrated that R2e_QSK from Gardnerella vaginalis modifies its active site tyrosine to DOPA, which is essential for activity.
• Determined the first high-resolution structures of R2e_QSK in both unmodified and DOPA-modified states.

Conclusions:

• R2e_QSK represents a significant functional variant within the metal-free RNR subclass.
• Tyrosine to DOPA modification in R2e_QSK is critical for its catalytic activity.
• The structural data provides insights into the mechanism of this unique radical generation pathway.