Probing the activity of NTHL1 orthologs by targeting conserved amino acid residues

•E. coli Asp44 plays a different role than its homologous residue in human NTHL1.•hNTHL1 Gln287 in involved in opposite base specificity as well as catalysis.•EcoNth Thr121 is involved in DNA binding and catalysis.•EcoNth and hNTHL1 demonstrate similar substrate specificity for DHT.