Structure and Biological Function of the RNA Pyrophosphohydrolase BdRppH from Bdellovibrio bacteriovorus

Messing, Simon; Gabelli, Sandra B.; Liu, Quansheng; Čelešnik, Helena; Belasco, Joel G.; Piñeiro, Silvia A.; Amzel, L. Mario

doi:10.1016/j.str.2008.12.022

Cited by 39 publications

(46 citation statements)

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“…A 43-nucleotide RNA (5Ј-GGAAUCUCUCUCUCUCUCUAUGCUCUCUCUCUCUCUCUC UCUC-3Ј) was transcribed and purified as previously described (1,23). To prepare the 5Ј-labeled substrate, phosphates on the 5Ј end were removed by Antarctic phosphatase (New England BioLabs) according to the manufacturer's instructions and rephosphorylated with [␥- To prepare the 3Ј-labeled RNA substrate, phosphates were removed from the 5Ј end with Antarctic phosphatase and the RNA was labeled using T4 RNA ligase (New England BioLabs) and [5Ј- 4C) and quenched with 95% formamide, 25 mM EDTA, 0.02% bromophenol blue, and 0.02% cyanol blue.…”

Section: Methodsmentioning

confidence: 99%

“…In addition to helping control the concentrations of crucial metabolites (e.g., ADP-ribose [12,19], UDP-glucose [46], and coenzyme A [4]), members of the superfamily also play key roles in biosynthetic pathways (e.g., folate biosynthesis [13]). It has been shown that some members of the Nudix family, i.e., Schizosaccharomyces pombe Dcp2 (31) and Escherichia coli and Bdellovibrio bacteriovorax RppH (7,23), may also play a role in RNA degradation by decapping mRNA.…”

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confidence: 99%

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The Nudix Hydrolase CDP-Chase, a CDP-Choline Pyrophosphatase, Is an Asymmetric Dimer with Two Distinct Enzymatic Activities

Duong‐Ly

Gabelli

et al. 2011

J Bacteriol

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A Nudix enzyme from Bacillus cereus (NCBI RefSeq accession no. NP_831800) catalyzes the hydrolysis of CDP-choline to produce CMP and phosphocholine. Here, we show that in addition, the enzyme has a 335 RNA exonuclease activity. The structure of the free enzyme, determined to a 1.8-Å resolution, shows that the enzyme is an asymmetric dimer. Each monomer consists of two domains, an N-terminal helical domain and a C-terminal Nudix domain. The N-terminal domain is placed relative to the C-terminal domain such as to result in an overall asymmetric arrangement with two distinct catalytic sites: one with an "enclosed" Nudix pyrophosphatase site and the other with a more open, less-defined cavity. Residues that may be important for determining the asymmetry are conserved among a group of uncharacterized Nudix enzymes from Gram-positive bacteria. Our data support a model where CDP-choline hydrolysis is catalyzed by the enclosed Nudix site and RNA exonuclease activity is catalyzed by the open site. CDPChase is the first identified member of a novel Nudix family in which structural asymmetry has a profound effect on the recognition of substrates.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

The Nudix Hydrolase CDP-Chase, a CDP-Choline Pyrophosphatase, Is an Asymmetric Dimer with Two Distinct Enzymatic Activities

Duong‐Ly

Gabelli

et al. 2011

J Bacteriol

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“…This family of enzymes has a characteristic fold consisting of two β sheets flanked by three α helices. Whereas E. coli RppH (EcRppH) and Bdellovibrio bacteriovorus RppH (BdRppH) catalyze the conversion of RNA 5′ triphosphate to 5′ monophosphate in a single step (i.e., with the release of pyrophosphate) (3,12), the B. subtilis enzyme performs this reaction in two steps, releasing two phosphate ions (4). Intrigued by this difference and conscious of the importance of B. subtilis as an alternative biological model for bacterial mRNA decay, we resolved the crystal structure of the B. subtilis RppH (BsRppH) enzyme in its free form, as well as that bound to GTP and a 5′-triphosphorylated dinucleotide RNA.…”

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confidence: 99%

Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates

Piton

Larue

Thillier

et al. 2013

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

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The initiation of mRNA degradation often requires deprotection of its 5′ end. In eukaryotes, the 5′-methylguanosine (cap) structure is principally removed by the Nudix family decapping enzyme Dcp2, yielding a 5′-monophosphorylated RNA that is a substrate for 5′ exoribonucleases. In bacteria, the 5′-triphosphate group of primary transcripts is also converted to a 5′ monophosphate by a Nudix protein called RNA pyrophosphohydrolase (RppH), allowing access to both endo-and 5′ exoribonucleases. Here we present the crystal structures of Bacillus subtilis RppH (BsRppH) bound to GTP and to a triphosphorylated dinucleotide RNA. In contrast to Bdellovibrio bacteriovorus RppH, which recognizes the first nucleotide of its RNA targets, the B. subtilis enzyme has a binding pocket that prefers guanosine residues in the second position of its substrates. The identification of sequence specificity for RppH in an internal position was a highly unexpected result. NMR chemical shift mapping in solution shows that at least three nucleotides are required for unambiguous binding of RNA. Biochemical assays of BsRppH on RNA substrates with single-base-mutation changes in the first four nucleotides confirm the importance of guanosine in position two for optimal enzyme activity. Our experiments highlight important structural and functional differences between BsRppH and the RNA deprotection enzymes of distantly related bacteria.RNA stability | 5′-processing | RNA decapping R NA turnover is a major target for the control of gene expression. Although the RNA maturation and degradation machineries of two of the best studied model bacteria, Escherichia coli and Bacillus subtilis, differ significantly (1, 2), they share an enzyme that deprotects the 5′ ends of primary transcripts by converting the 5′-triphosphate group to a 5′ monophosphate (3, 4). The 5′ monophosphorylated RNA is a much better substrate for the major endoribonuclease RNase E in E. coli (5) and for the 5′-3′ exoribonuclease RNase J1 in B. subtilis (6). The structural basis for this preference is known in both cases. Binding of a 5′ monophosphate to a specific pocket stimulates the efficiency of RNase E cleavage at downstream sites, through a conformational change in the enzyme (7), whereas in the case of RNase J1, a 5′ triphosphate is thought to reduce enzyme activity because the distance between the 5′-phosphate binding pocket and the active site is optimized for a nucleoside 5′ monophosphate (8). The primary endoribonuclease of B. subtilis, RNase Y, has also been shown to prefer the 5′-monophosphorylated version of at least one RNA, the yitJ riboswitch, but the molecular basis for this preference is not yet known (9).The enzyme responsible for RNA deprotection in both E. coli and B. subtilis is RNA pyrophosphohydrolase (RppH). This enzyme is a member of the very ancient family of nucleoside diphosphate linked to X (Nudix) hydrolases, involved in a wide range of important biological reactions, including the hydrolysis of ADP ribose, 8-oxoguanosine, and AppppA (10). Removal of...

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“…The Nudix motif (23 residues from Gly-38 i.e. the MutT signature (GX 5 EX 7 REUXEEXGU), adopts the characteristic strand-loop-helix-loop (SLHL) structure formed by ␤-3Ј, L-B, ␣-1, and L-C (39,40). The crystal of the apo form contains two protein molecules per asymmetric unit, and they are very similar to each other with root mean square deviation (r.m.s.d.)…”

Section: Resultsmentioning

confidence: 99%

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

Nakamura

Meshitsuka

Kitagawa

et al. 2010

Journal of Biological Chemistry

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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 ...

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Structure and Biological Function of the RNA Pyrophosphohydrolase BdRppH from Bdellovibrio bacteriovorus

Cited by 39 publications

References 50 publications

The Nudix Hydrolase CDP-Chase, a CDP-Choline Pyrophosphatase, Is an Asymmetric Dimer with Two Distinct Enzymatic Activities

The Nudix Hydrolase CDP-Chase, a CDP-Choline Pyrophosphatase, Is an Asymmetric Dimer with Two Distinct Enzymatic Activities

Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates

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

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