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

Kirillov S. Isupov M. Paterson N.G. Wiener R. Abeldenov S. Saper M.A. Rouvinski A.
7 January 2025 American Chemical Society

Biochemistry
2025 #64 Issue 1 67 - 82 pp.

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.

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Department of General Biology and Genomics, L. N. Gumilyov Eurasian National University, Astana, 010008, Kazakhstan
Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada, The Hebrew University of Jerusalem, Jerusalem, 9112102, Israel
National Center for Biotechnology, Astana, 010000, Kazakhstan
Biosciences, University of Exeter, Exeter, EX4 4QD, United Kingdom
Diamond Light Source, Oxfordshire, OX11 0DE, United Kingdom
Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew University of Jerusalem, Jerusalem, 9112102, Israel
Department of Biological Chemistry, University of Michigan, Ann Arbor, 48109, MI, United States
The Kuvin Center for the Study of Infectious and Tropical Diseases, The Hebrew University of Jerusalem, Jerusalem, 9112102, Israel

Department of General Biology and Genomics
Department of Microbiology and Molecular Genetics
National Center for Biotechnology
Biosciences
Diamond Light Source
Department of Biochemistry and Molecular Biology
Department of Biological Chemistry
The Kuvin Center for the Study of Infectious and Tropical Diseases

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