A Structurally Divergent Class Ia Ribonucleotide Reductase from a Tick-Borne Pathogen.

Area of Science:

• Biochemistry and Molecular Biology
• Enzymology
• Structural Biology

Background:

• Ribonucleotide reductases (RNRs) are essential enzymes for DNA biosynthesis, catalyzing the production of 2'-deoxynucleotides.
• Class I RNRs utilize a β subunit to generate a crucial thiyl radical in the α subunit, initiating the catalytic cycle.
• The oxidant in the β subunit can be a tyrosyl radical (Y•), a dimetal cluster, or a dihydroxylphenylalanine (DOPA) radical, with metal dependence varying across RNR classes.

Purpose of the Study:

• To characterize the structure and function of the β subunit from *Francisella hispaniensis* (Fh) RNR, an enzyme from an uncharacterized sequence cluster found in human pathogens.
• To investigate the nature of the cofactor and unusual structural features of *Fh* β and their potential implications for the bacterium's survival.

Main Methods:

• Structural analysis of the apo form of *Fh* β.
• Biochemical assays to determine cofactor composition (Fe₂(III/III)/Y^•).
• Enzyme kinetics and radical stability studies, including hydroxyurea sensitivity assays during turnover with the α subunit.

Main Results:

• The *Fh* β subunit harbors a Fe₂(III/III)/Y^• cofactor, similar to eukaryotic and *E. coli* class Ia RNRs.
• Unusual structural features were observed, including an unwound helix in the apo form and a repositioned tyrosyl radical.
• The tyrosyl radical in resting *Fh* β is highly resistant to reduction but becomes significantly more sensitive during catalytic turnover with the α subunit.

Conclusions:

• The *Fh* RNR β subunit possesses unique structural and functional properties, including a conserved aromatic residue (W194) near W37, potentially involved in α-Cys oxidation.
• The radical's modulated sensitivity to reduction suggests an adaptation to evade host redox defenses targeting the pathogen's essential RNR.
• These findings provide insights into the evolutionary adaptations of RNRs in pathogenic bacteria and their interaction with host environments.