An unconventional family 1 uracil DNA glycosylase in Nitratifractor salsuginis

Abstract

The uracil DNA glycosylase superfamily consists of at least six families with a diverse specificity towards DNA base damage. Family 1 UNG exhibits exclusive specificity on uracil-containing DNA. Here, we report a family 1 UNG homolog from Nitratifractor salsuginis with distinct biochemical features that differentiate it from conventional family 1 UNGs. Globally, the crystal structure of N. salsuginis UNG shows a few additional secondary structural elements. Biochemical and enzyme kinetic analysis, coupled with structural determination, molecular modeling and molecular dynamics simulations, shows that N. salsuginis UNG contains a salt bridge network that plays an important role in DNA backbone interactions. Disruption of the amino acid residues involved in the salt bridges greatly impedes the enzymatic activity. A tyrosine residue in motif 1 (GQDPY) is one of the distinct sequence features setting family 1 UNG apart from other families. The crystal structure of Y81G mutant indicates that several subtle changes may account for its inactivity. Unlike the conventional family 1 UNG enzymes, N. salsuginis UNG is not inhibited by Ugi, a potent inhibitor specific for family 1 UNG. This study underscores the diversity of paths that a uracil DNA glycosylase may take to acquire its unique structural and biochemical properties during evolution.

Targeting Uracil-DNA Glycosylases for Therapeutic Outcomes Using Insights from Virus Evolution

“…New variants of almost all UDG families are regularly reported in the literature [30,31], including variants of family 1 enzymes (i.e. Ungs) [32,33]. Ung variants from pathogens may have relevance as targets for therapeutics development to combat pathogenesis [26][27][28][34][35][36][37], and in some cases novel chemical entities of interest have been reported [10,11,14,28,38] (Table 1).…”

“…In pathogen encoded Ungs, structures are sufficiently diverged in important catalytic respects, that their selective inhibition might present more options for drug design. For example, the variants encoded by g-herpesviruses are sufficiently different in important respects with regard to catalysis as to present unique possibilities for their selective inhibition (Table 1, Figure 1, Figure 2) [33,34]. Another example of a significantly variant Ung, is that encoded by vaccinia virus.…”

“…EBV,KSHV) [59][60][61][62] Specialised viral Ung in lytic phase, and utilisation of UNG2 in the latent phase maintenance/reactivation. [3,33,34] Viral Ung is implicated in latent maintenance in HSV-1 [61], while in hCMV and g-herpesviruses viral Ung is an essential part of the lytic phase replication machinery [34,60,61]. In the latter class, UNG2 is recruited to assist in latency-associated processes [62].…”

“…In the g-herpesviruses, Epstein-Barr (EBV) [HHV-4] and Kaposi's sarcoma-associated herpesvirus (KSHV) , the pre-catalytic loop of Ung is seen to be appended by a sequence motif of conserved length but variable composition ( Figure 2). Structural studies reveal that upon DNA docking this elaborated motif adopts a conserved structure [33,34] ( Figure 1). In engineered mutant forms of g-herpesvirus Ung, lacking the loop motif appendage, infecting viruses are highly compromised in lytic phase replication [60,92] and equivalent mutants are rendered biophysically unstable [34].…”

“…Interestingly, although it has already been effectively targeted via its PPI with viral polymerase E9, the poxvirus Ung has a structurally diverged loop, among other gross differences relating to its essential role in viral replication [26]. The bherpesvirus Ungs feature a charged apical pre-catalytic loop residue; g-herpesvirus Ungs have a separate character to take advantage of in this pre-catalytic loop area, by virtue of a structured extension to that loop, necessary for effective lytic replication [33,34]. In terms of designing a drug, this has a slight complication in that the loop is disordered prior to DNA docking; nevertheless, the structural and mutagenesis data available provide important design clues [34].…”