Octahedral Iron in Catalytic Sites of Endonuclease IV from Staphylococcus aureus and Escherichia coli

Saveliy Kirillov, Michail Isupov, Neil G. Paterson, Reuven Wiener, Sailau Abeldenov, Mark A. Saper*, Alexander Rouvinski*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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 Zn2+. The 1.05 Å structure of Nfo from the Gram-positive organism S. aureus (SaNfo) revealed two inner Fe2+ ions and one Zn2+ as confirmed by dispersive anomalous difference maps. SaNfo has a previously undescribed water molecule liganded to Fe1 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 Zn2+ at the corresponding site. The 1.9 Å structure of E. coli Nfo (EcNfo), purified without added metals, revealed that metal 2 is Fe2+ and not Zn2+. Octahedral coordination for the sites occupied by Fe2+ suggests a stereoselective mechanism for differentiating between Fe2+ and Zn2+ in this enzyme class. Kinetics and an inhibitor competition assay of SaNfo reveal product inhibition (or slow product release), especially at low ionic strength, caused in part by a Lys-rich DNA binding loop present in SaNfo and Gram-positive species but not in EcNfo. 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.

Original languageEnglish
Pages (from-to)67-82
Number of pages16
JournalBiochemistry
Volume64
Issue number1
DOIs
StatePublished - 7 Jan 2025

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