The structure of uracil-DNA glycosylase from Atlantic cod (Gadus morhua) reveals cold-adaptation features

Abstract

Uracil-DNA glycosylase (UDG; EC 3.2.2.3) is a DNA-repair protein that catalyses the hydrolysis of promutagenic uracil residues from single- or double-stranded DNA, generating free uracil and abasic DNA. The crystal structure of the catalytic domain of cod uracil-DNA glycosylase (cUDG) has been determined to 1.9 A resolution, with final R factors of 18.61 and 20.57% for the working and test sets of reflections, respectively. This is the first crystal structure of a uracil-DNA glycosylase from a cold-adapted species and a detailed comparison with the human enzyme is performed in order to rationalize the cold-adapted behaviour of the cod enzyme at the structural level. The catalytic domain of cUDG comprises 223 residues, with a sequence identity to the human UDG of 75%. The tertiary structures of the two enzymes are also similar, with an overall displacement in main-chain atomic positions of 0.63 A. The amino-acid substitutions and the differences in intramolecular hydrogen bonds, hydrophobic interactions, ion-pair interactions and electrostatic potentials are compared and discussed in order to gain insight into the factors that cause the increased activity and reduced thermostability of the cod enzyme. In particular, the reduced number of strong ion-pair interactions in the C-terminal half of cUDG is believed to greatly affect the flexibility and/or stability. Increased positive electrostatic surface potential on the DNA-facing side of cUDG seems to be responsible for increasing the affinity for the negatively charged DNA compared with that of hUDG.

Reduced Hydrophobicity of the Minor Groove Intercalation Loop is Critical for Efficient Catalysis by Cold Adapted Uracil-DNA N-Glycosylase from Atlantic Cod

“…The catalytic domain of cod UNG (cUNG) has been produced (in E. coli) and purified, and characterisation experiments shows that the activity of cUNG is more thermolabile and has a 10-fold increase in catalytic efficiency at optimum conditions (pH 7.0 and 50 mM NaCl) compared to hUNG [34]. The crystal structure of the catalytic domain of cUNG has been determined at 1.9 Å resolution and a comparative analysis of the structures of cUNG and hUNG suggested that a less stable C-terminal part and an increased electrostatic surface potential near the active site might be responsible for the cold adapted behaviour of the cod enzyme [6]. The importance of an optimised positive electrostatic surface potential for the cold adapted features of cUNG has been confirmed experimentally by mutational and structural analysis [35], and theoretical calculation studies have suggested that cUNG in part uses local flexibility of the minor groove intercalation loop (leucine-loop) as a strategy for cold adaptation [6,36].…”

“…The crystal structure of the catalytic domain of cUNG has been determined at 1.9 Å resolution and a comparative analysis of the structures of cUNG and hUNG suggested that a less stable C-terminal part and an increased electrostatic surface potential near the active site might be responsible for the cold adapted behaviour of the cod enzyme [6]. The importance of an optimised positive electrostatic surface potential for the cold adapted features of cUNG has been confirmed experimentally by mutational and structural analysis [35], and theoretical calculation studies have suggested that cUNG in part uses local flexibility of the minor groove intercalation loop (leucine-loop) as a strategy for cold adaptation [6,36]. Recently the first thermal stability analysis of cUNG and hUNG, performed by DSC, was published and shows that the thermal melting temperature (T m ) of cUNG is 9°C lower than for hUNG [37].…”

“…Uracil in DNA may result from deamination of cytosine [1] or misincorporation during replication [2]. The crystal structure of the catalytic domain is known from a wide range of organisms including human [3], herpes simplex virus 1 (HSV-1) [4], E. coli [5], Atlantic cod [6], D. radiodurans [7], Vibrio cholerae [8], Vaccinia virus [9], Mycobacterium tuberculosis [10], Bacillus substilis (Banos-Sanz, 2013) and Staphylococcus aureus [11]. All ten enzymes have similar structures and consist of a classic single domain α/β-fold with a central four stranded parallel and twisted β-sheet surrounded by eight to eleven α-helices.…”

Increased Flexibility as a Strategy for Cold Adaptation

“…This enzyme, which is the first in the base excision repair pathway, catalyzes the hydrolysis of promutagenic uracil residues from single-or double-stranded DNA. The crystal structure of the catalytic domain of UDG from several species are known: human (hUDG) (14), cod (cUDG) (15), virus 1 (16), and Escherichia coli (17). The threedimensional structure of UDG in complex with DNA has also been determined (16, 18 -21).…”

“…The amino acids at positions 171 and 275 seem to be two key residues when explaining the difference in the surface potential (15,24). Several mutants were subsequently constructed and analyzed in terms of kinetic, thermodynamic, and structural properties (12,15,24).…”