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

Abstract

The base excision repair DNA glycosylases, EcoNth and hNTHL1, are homologous, with reported overlapping yet different substrate specificities. The catalytic amino acid residues are known and are identical between the two enzymes although the exact structures of the substrate binding pockets remain to be determined. We sought to explore the sequence basis of substrate differences using a phylogeny-based design of site-directed mutations. Mutations were made for each enzyme in the vicinity of the active site and we examined these variants for glycosylase and lyase activity. Single turnover kinetics were done on a subgroup of these, comparing activity on two lesions, 5,6-dihydrouracil and 5,6-dihydrothymine, with different opposite bases. We report that wild type hNTHL1 and EcoNth are remarkably alike with respect to the specificity of the glycosylase reaction, and although hNTHL1 is a much slower enzyme than EcoNth, the tighter binding of hNTHL1 compensates, resulting in similar kcat/Kd values for both enzymes with each of the substrates tested. For the hNTHL1 variant Gln287Ala, the specificity for substrates positioned opposite G is lost, but not that of substrates positioned opposite A, suggesting a discrimination role for this residue. The EcoNth Thr121 residue influences enzyme binding to DNA, as binding is significantly reduced with the Thr121Ala variant. Finally, we present evidence that hNTHL1 Asp144, unlike the analogous EcoNth residue Asp44, may be involved in resolving the glycosylase transition state.

Caught in motion: human NTHL1 undergoes interdomain rearrangement necessary for catalysis

“…In the open state, the strictly conserved catalytic residues, Lys 220 and Asp 239, which are located ∼5 Å apart in the bacterial enzymes, are found ∼23 Å apart (Figure 1 ). Therefore, hNTHL1 must undergo a conformational change in order for catalysis to occur ( 17 , 19 , 59–61 ). This finding was unexpected as the bacterial homologs were captured in a similar closed conformation, in the presence and absence of DNA (Figure 1 ).…”

“…Robey-Bond et al. investigated several residues which were deemed important for catalysis in E. coli and yet had a different amino acid residue at the analogous position in mammalian NTHL1 ( 19 ), including Asn279, Gly280, and Gln287 ( 19 ). Our crystal structure of hNTHL1 shows that Asn 279 and Gly 280 are located near linker 2, with the former contacting the backbone carbonyl of Ala 137 in that linker.…”

“…hNTHL1 exhibits a low affinity for undamaged DNA (30 μM ( 79 ) vs . nM range for lesion-containing DNA ( 19 )), therefore we can assume that there is little appreciable DNA binding signal in the undamaged DNA curve. hNTHL1 binds to DNA containing THF but, as this lesion is non-cleavable, the enzyme cannot cycle through catalysis, thus keeping the enzyme in a lesion-bound state.…”

Caught in Motion: Human NTHL1 Undergoes Interdomain Rearrangement Necessary for Catalysis

“…In the open state, the strictly conserved catalytic residues, Lys 220 and Asp 239, which are located ~5 Å apart in the bacterial enzymes, are found ~23 Å apart (Figure 1). Therefore, hNTHL1 must undergo a conformational change in order for catalysis to occur (17,19,(60)(61)(62). This finding was unexpected as the bacterial homologs were captured in a similar closed conformation, in the presence and absence of DNA (Figure 1).…”

“…Nth is a bifunctional DNA glycosylase with a marked preference for oxidized pyrimidine substrates (16). While both Escherichia coli Nth (EcoNth) and human NTHL1 (hNTHL1) glycosylases display a preference for excising lesions opposite G, the human enzyme is slower than the bacterial enzyme on most substrates (17)(18)(19). The first bacterial Nth crystal structure, EcoNth, revealed that the enzyme consists of two globular α -helical domains, a six-helical bundle domain and a [4Fe4S] cluster domain.…”