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 ...

Microbial production of cis,cis-muconate (ccMA) from phenolic compounds obtained by chemical depolymerization of lignin is a promising approach to valorize lignin. Because microbial production requires a large amount of carbon and energy source, it is desirable to establish a ccMA-producing strain that utilizes lignin-derived phenols instead of general sources like glucose. We isolated Pseudomonas sp. strain NGC7 that grows well on various phenolic compounds derived from p-hydroxyphenyl, guaiacyl, and syringyl units of lignin. An NGC7 mutant of protocatechuate (PCA) 3,4-dioxygenase and ccMA cycloisomerase genes (NGC703) lost the ability to grow on vanillate and p-hydroxybenzoate but grew normally on syringate. Introduction of a plasmid carrying genes encoding PCA decarboxylase, flavin prenyltransferase, vanillate O-demethylase, and catechol 1,2-dioxygenase into NGC703 enabled production of 3.2 g/L ccMA from vanillate with a yield of 75% while growing on syringate. This strain also produced ccMA from birch lignin-derived phenols. All these results indicate the utility of NGC7 in glucose-free ccMA production.

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