Structure of a Coenzyme A Pyrophosphatase from
            <i>Deinococcus radiodurans</i>
            : a Member of the Nudix Family

Kang, Lin Woo; Gabelli, Sandra B.; Bianchet, M.A.; Xu, Wenyue; Bessman, Maurice J.; Amzel, L. Mario

doi:10.1128/jb.185.14.4110-4118.2003

Cited by 36 publications

(45 citation statements)

References 40 publications

(36 reference statements)

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“…1). Sequence subunit analysis did not suggest that these proteins were members of any of the known subfamilies of Nudix hydrolases (6,13,28). The mutT1 M. tuberculosis knockout mutant (MT1K) was isolated from the transposon library described previously (12).…”

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confidence: 99%

Identification of Nudix Hydrolase Family Members with an Antimutator Role in Mycobacterium tuberculosis and Mycobacterium smegmatis

et al. 2006

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Mycobacterium tuberculosis and Mycobacterium smegmatis MutT1, MutT2, MutT3, and Rv3908 (MutT4) enzymes were screened for an antimutator role. Results indicate that both MutT1, in M. tuberculosis and M. smegmatis, and MutT4, in M. smegmatis, have that role. Furthermore, an 8-oxo-guanosine triphosphatase function for MutT1 and MutT2 is suggested.Oxidized guanine (8-oxo-G) is a potent mutagen because of its ambiguous pairing with cytosine and adenine. The Escherichia coli MutT protein specifically hydrolyzes both 8-oxo-deoxyguanosine triphosphate (8-oxo-dGTP) and 8-oxo-guanosine triphosphate (8-oxo-rGTP), preventing their misincorporation in DNA and RNA opposite template A (10,23,24,26). The MutT E. coli protein has an antimutator function, and it was the first enzyme of the MutT/Nudix hydrolase family, characterized by a 23-amino-acid region, to be studied. Nudix hydrolases, are so named because the ones characterized so far all hydrolyze a nucleoside diphosphate linked to some other moiety, X. Besides oxidized guanine, they were shown to degrade other substrates, such as NADH, GDP-mannose, or ADPribose (2,3,8,9,14,15,18).Here we have investigated the role of the putative Nudix hydrolases MutT1, MutT2, MutT3, and Rv3908 (MutT4) of M. tuberculosis (5) and their putative M. smegmatis homologues (sequences were obtained from The Institute for Genomic Research [TIGR] website; www.tigr.org) as antimutators (Fig. 1). Sequence subunit analysis did not suggest that these proteins were members of any of the known subfamilies of Nudix hydrolases (6, 13, 28). The mutT1 M. tuberculosis knockout mutant (MT1K) was isolated from the transposon library described previously (12). Selection was done by plating aliquots of each independent insertional mutant onto 7H10 plates containing rifampin (Rif) at 2 g/ml, two times the MIC of Rif for M. tuberculosis used by Morlock et al. (17). One of the clones giving a higher number of Rif-resistant (Rif r ) colonies than the wild-type strain harbored an insertion in mutT1. All other mutants were generated by allelic replacement using a replication temperature-sensitive vector harboring the counterselectable marker sacB to carry a kanamycin cassette-disrupted copy of the genes of interest (20). The bacterial strains, plasmids, and primers employed in this study are provided in the supplemental material. DNA isolation, cloning, and Southern hybridization were performed according to standard techniques. Complemented strains of the M. smegmatis mutants were generated by electroporation of the pVV16-derived vectors (11) described in the supplemental material. In 20 independent experiments, we carried out an adapted LuriaDelbruck fluctuation test, as described by Morlock et al. (17). The results obtained are summarized in Table 1. MutT1 deficiency in M. tuberculosis resulted in a 15.5-fold spontaneous mutation frequency increase by rifampin resistance screening compared with the wild-type strain. A similar 12-fold increase was observed for the mutT1 mutant of M. smegmatis. Furthermore, we observe...

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confidence: 99%

Identification of Nudix Hydrolase Family Members with an Antimutator Role in Mycobacterium tuberculosis and Mycobacterium smegmatis

et al. 2006

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“…CAD18986) bears sequence similarity to the Nudix hydrolase family, showing 38% sequence similarity to a CoA pyrophosphatase from Deinococcus radiodurans (22). This family of enzymes hydrolyzes diphosphates of various nucleotide-containing molecules.…”

Section: Resultsmentioning

confidence: 99%

“…This family of enzymes hydrolyzes diphosphates of various nucleotide-containing molecules. Nudix hydrolases are characterized by a conserved Nudix box sequence and, in the case of Nudix enzymes, by using CoA as a substrate, a conserved NuCoA motif present directly upstream of the Nudix box (22). ThnR possesses slight variations of both motifs characteristic of a CoA pyrophosphatase and was thought to be the enzyme responsible for processing CoA or a CoA-containing precursor of thienamycin (Fig.…”

Section: Resultsmentioning

confidence: 99%

Four enzymes define the incorporation of coenzyme A in thienamycin biosynthesis

Freeman¹,

Moshos

Bodner

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The enzymatic activities of three proteins encoded by the thienamycin gene cluster of Streptomyces cattleya (ThnR, ThnH, and ThnT) have been shown to incrementally cleave CoA to afford the active side-chain component of the ␤-lactam antibiotic thienamycin. These results supersede proposals based on earlier radiochemical incorporation experiments. For 20 years it has been thought that cysteine was directly incorporated into the antibiotic. Specific, stepwise truncation of CoA to 4-phosphopantetheine, pantetheine, and finally cysteamine was observed with ThnR, ThnH, and ThnT, respectively, in a series of coupled enzymatic assays. Pantetheinylated carbapenams were synthesized to address possible thienamycin biosynthetic intermediates and were shown to be effective substrates for the pantetheine-cleaving enzyme ThnT. Finally, a fourth gene, thnF, was shown to encode a protein capable of N-acetylating a model compound containing cysteamine in the presence of acetyl-CoA, consistent with the production of the S. cattleya cometabolite, N-acetylthienamycin. Taken together, these four enzymes are proposed to siphon CoA from primary metabolism to create the side chains for the predominant S. cattleya carbapenems, thienamycin and N-acetylthienamycin, in a process likely to be general for the broader class of these antibiotics.␤-lactam antibiotics ͉ carbapenem ͉ pyrophosphatase ͉ acylase ͉ phosphatase

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“…Both DR1183 and DR1022 neighbor a Nudix hydrolase gene each (43,44). Deducing possible functions based on genomic context has led to the proposal that MazG(-like) domains may be involved in non-canonical NTP processing.…”

Section: Discussionmentioning

confidence: 99%

“…The uracyl ring is settled between hydrophobic residues Phe 17 and Leu 43 , each one making van der Waals interactions on both sides of the moiety. Furthermore, O2, N3, and O4 of the uracyl moiety interact with three water molecules, with W3, W4, and W5, respectively (Fig.…”

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confidence: 99%

Structural and Functional Insights into DR2231 Protein, the MazG-like Nucleoside Triphosphate Pyrophosphohydrolase from Deinococcus radiodurans

Gonçalves¹,

Sanctis

McSweeney

2011

Journal of Biological Chemistry

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Deinococcus radiodurans is among the very few bacterial species extremely resistant to ionizing radiation, UV light, oxidizing agents, and cycles of prolonged desiccation. The proteome of D. radiodurans reflects the evolutionary pressure exerted by chronic exposure to (nonradioactive) forms of DNA and protein damage. A clear example of this adaptation is the overrepresentation of protein families involved in the removal of non-canonical nucleoside triphosphates (NTPs) whose incorporation into nascent DNA would promote mutagenesis and DNA damage. The three-dimensional structure of the DR2231 protein has been solved at 1.80 Å resolution. This protein had been classified as an all-␣-helical MazG-like protein. The present study confirms that it holds the basic structural module characteristic of the MazG superfamily; two helices form a rigid domain, and two helices form a mobile domain and connecting loops. Contrary to what is known of MazG proteins, DR2231 protein shows a functional affinity with dUTPases. Enzymatic and isothermal calorimetry assays have demonstrated high specificity toward dUTP but an inability to hydrolyze dTTP, a typical feature of dUTPases. Co-crystallization with the product of hydrolysis, dUMP, in the presence of magnesium or manganese cations, suggests similarities with the dUTP/dUDP hydrolysis mechanism reported for dimeric dUTPases. The genome of D. radiodurans encodes for all enzymes required for dTTP synthesis from dCMP, thus bypassing the need of a dUTPase. We postulate that DR2231 protein is not essential to D. radiodurans and rather performs "house-cleaning" functions within the framework of oxidative stress response. We further propose DR2231 protein as an evolutionary precursor of dimeric dUTPases.

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Structure of a Coenzyme A Pyrophosphatase from Deinococcus radiodurans : a Member of the Nudix Family

Cited by 36 publications

References 40 publications

Identification of Nudix Hydrolase Family Members with an Antimutator Role in Mycobacterium tuberculosis and Mycobacterium smegmatis

Identification of Nudix Hydrolase Family Members with an Antimutator Role in Mycobacterium tuberculosis and Mycobacterium smegmatis

Four enzymes define the incorporation of coenzyme A in thienamycin biosynthesis

Structural and Functional Insights into DR2231 Protein, the MazG-like Nucleoside Triphosphate Pyrophosphohydrolase from Deinococcus radiodurans

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