Abstract
During Staphylococcus aureus infections, reactive oxygen species cause DNA damage, including nucleotide base modification. After removal of the defective base, excision repair requires an endonuclease IV (Nfo), which hydrolyzes the phosphodiester bond 5′ to the abasic nucleotide. This class of enzymes, typified by the enzyme from Escherichia coli, contains a catalytic site with three metal ions, previously reported to be all Zn 2+ . The 1.05 Å structure of Nfo from the Gram-positive organism S. aureus ( Sa Nfo) revealed two inner Fe 2+ ions and one Zn 2+ as confirmed by dispersive anomalous difference maps. Sa Nfo has a previously undescribed water molecule liganded to Fe 1 forming an octahedral coordination geometry and hydrogen bonded to Tyr33, an active site residue conserved in many Gram-positive bacteria, but which is Phe in Gram-negative species that coordinate Zn 2+ at the corresponding site. The 1.9 Å structure of E. coli Nfo ( Ec Nfo), purified without added metals, revealed that metal 2 is Fe 2+ and not Zn 2+ . Octahedral coordination for the sites occupied by Fe 2+ suggests a stereoselective mechanism for differentiating between Fe 2+ and Zn 2+ in this enzyme class. Kinetics and an inhibitor competition assay of Sa Nfo reveal product inhibition (or slow product release), especially at low ionic strength, caused in part by a Lys-rich DNA binding loop present in Sa Nfo and Gram-positive species but not in Ec Nfo. Biological significance of the slow product release is discussed. Catalytic activity in vitro is optimal at 300 mM NaCl, which is consistent with the halotolerant phenotype of S. aureus .