Structural and Dynamic Features of the MutT Protein in the Recognition of Nucleotides with the Mutagenic 8-Oxoguanine Base

Abstract

Escherichia coli MutT hydrolyzes 8-oxo-dGTP to 8-oxodGMP, an event that can prevent the misincorporation of 8-oxoguanine opposite adenine in DNA. Of the several enzymes that recognize 8-oxoguanine, MutT exhibits high substrate specificity for 8-oxoguanine nucleotides; however, the structural basis for this specificity is unknown. The crystal structures of MutT in the apo and holo forms and in the binary and ternary forms complexed with the product 8-oxodGMP and 8-oxo-dGMP plus Mn 2؉ , respectively, were determined. MutT strictly recognizes the overall conformation of 8-oxo-dGMP through a number of hydrogen bonds. This recognition mode revealed that 8-oxoguanine nucleotides are discriminated from guanine nucleotides by not only the hydrogen bond between the N7-H and O␦ (N119) atoms but also by the syn glycosidic conformation that 8-oxoguanine nucleotides prefer. Nevertheless, these discrimination factors cannot by themselves explain the roughly 34,000-fold difference between the affinity of MutT for 8-oxo-dGMP and dGMP. When the binary complex of MutT with 8-oxo-dGMP is compared with the ligand-free form, ordering and considerable movement of the flexible loops surrounding 8-oxodGMP in the binary complex are observed. These results indicate that MutT specifically recognizes 8-oxoguanine nucleotides by the ligand-induced conformational change.Although spontaneous mutations are indispensable to the evolutionary process of living organisms, they can also be lethal to the organism. Among the various modified bases in DNA, RNA, and nucleotides, 8-oxoguanine (8-oxoG), 2 a damaged form of guanine (G) generated by reactive oxygen species, is known to have highly mutagenic potency because of its mispairing with adenine. Therefore, organisms have an error avoidance pathway for preventing mutations caused by 8-oxoG. The Escherichia coli MutT protein (129 amino acids, M r ϭ 14,900) hydrolyzes 8-oxo-dGTP and 8-oxo-GTP to their corresponding nucleoside monophosphates and inorganic pyrophosphate in the presence of Mg 2ϩ (1, 2). Because 8-oxodGTP and 8-oxo-GTP can be misincorporated opposite adenine by DNA and RNA polymerases, the hydrolysis of the damaged nucleotides by MutT can avoid replicational and transcriptional errors. In DNA, 8-oxoG paired with cytosine is excised by MutM, an 8-oxoG DNA glycosylase, whereas MutY, an adenine DNA glycosylase, removes adenine paired with 8-oxoG (3-6).The substrate specificities of enzymes that recognize 8-oxoG are quite varied. MutT exhibits high substrate specificity for 8-oxoG nucleotides; that is, the K m for 8-oxo-dGTP is 14,000-fold lower than that for dGTP (7). In contrast, human MutT homologue 1 (hMTH1) hydrolyzes not only 8-oxo-dGTP but also several oxidized purine nucleotides such as 2-oxo-dATP, 2-oxo-ATP, 8-oxo-dATP, and 8-oxo-ATP. In terms of the hydrolysis of 8-oxo-dGTP, the K m of hMTH1 for 8-oxo-dGTP is only 17-fold lower than that for dGTP (8, 9). The solution structure of hMTH1 as determined by NMR has revealed its overall architecture and possible substrate-binding region ...

Human MTH3 (NUDT18) Protein Hydrolyzes Oxidized Forms of Guanosine and Deoxyguanosine Diphosphates

“…Furthermore, the 8-oxoGua base-binding mode and the glycosidic conformation are quite different in MutT and MTH1, although the sugar and phosphate positions of the two 8-oxo-dGMPs are similar. In contrast to the finding that MutT and MTH1 exist as monomers (32,34,36), NUDT5 forms a homodimer with substantial domain swapping (33,35). In NUDT5, the nucleoside moiety of 8-oxo-dGDP binds to a completely different region near the dimer interface without ligand-induced conformational changes.…”

“…The substrate recognition and binding may be achieved by a few amino acid residues located near or within the active center, and thus, elucidation of the three-dimensional structures is essential for solving this problem. Analyses of the crystal structures of MutT, MTH1, and NUDT5, all of which are complexed with 8-oxo-Gua-containing nucleotides, have been performed (32)(33)(34)(35)(36)(37). In the cases of MutT and MTH1, the reaction product 8-oxo-dGMP binds to the same substrate- binding pocket composed of two ␤-sheets and one ␣-helix.…”

Enhanced resolution of molecular recognition to distinguish structurally similar molecules by different conformational responses of a protein upon ligand binding

“…The Xray structures are available for substrate-free MutT (3A6S) and for the complex with 8-oxo-dGMP (3A6T) (Nakamura et al, 2004(Nakamura et al, , 2010. Comparison of the substrate-free and 8-oxo-dGMP-bound structures reveals a remarkable difference.…”

Oxidative Damage to RNA and Expression Patterns of MTH1 in the Hippocampi of Senescence-Accelerated SAMP8 Mice and Alzheimer’s Disease Patients

“…To prevent such outcomes, organisms are equipped with molecular mechanisms to eliminate oxidized nucleotides from the nucleic acid precursor pools. The MutT protein of Escherichia coli is representative of this type of enzyme, and its mode of action on oxidized nucleotides has been studied extensively [8,9]. In mammalian cells, three types of MutT-related enzymes are known, and each exhibits distinct substrate specificities.…”