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

Duong‐Ly, Krisna C.; Gabelli, Sandra B.; Xu, Wenyue; Dunn, Christopher; Schoeffield, Andrew; Bessman, Maurice J.; Amzel, L. Mario

doi:10.1128/jb.00089-11

Cited by 10 publications

(18 citation statements)

References 48 publications

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“…These results indicate that UDP-Xase degrades RNA in the 3′ 5′ direction. This activity is similar to that of the B. cereus CDP-Chase [5].…”

Section: Resultssupporting

confidence: 78%

“…Glu162 is in the loop that connects strands β5 and β6 (residues 153–162). In the E162A mutant, residue 162 does not coordinate the divalent cation and the loop may move to a position farther away from the rest of the structure, as observed in other Nudix enzymes [5]. The R g and D max calculated from the wild-type UDP-Xase crystal structure are 23.9 Å and 65.6 Å, respectively; the R g and D max calculated from the SAXS data of the mutants show that E113A has a conformation similar to that observed in the crystal structure of the wild-type (Table 3; R g = 23.9 Å, D max = 65.0 Å).…”

Section: Resultsmentioning

confidence: 87%

“…The overall structure of the C-terminal catalytic domain is similar to that of other Nudix hydrolases but is most similar to that of human NUDT18 (PDB ID 3GG6; unpublished). Other Nudix hydrolases that share this fold include B. cereus CDP-Chase [5], Bdellovibrio bacteriovorax BdRppH [23], E. coli DHNTPase [38], and E. coli GDPMK [39].…”

Section: Resultsmentioning

confidence: 99%

“…Preparation of 5′- and 3′- radiolabeled RNA substrates and assays of RNase activity were carried out as described earlier [5], [23], [24]. To determine the presence of RNase activity and the directionality of this activity, reactions were carried out in 50 mM Tris pH 8.25, 5 mM MgCl 2 , 1 mM DTT, and 0.25 µM UDP-Xase with 40 nM 5′- or 3′-end labeled RNA; water was used in place of enzyme for the negative controls.…”

Section: Methodsmentioning

confidence: 99%

“…Based on its similarity with the CDP-Chase from Bacillus cereus, a Nudix enzyme that hydrolyzes CDP-choline and has, in addition, an RNA exonuclease activity [5], we tested UDP-Xase against RNA substrates and showed that it indeed has a 3′5′ exonuclease activity. The UDP-Xase from Streptococcus pneumoniae , may be a member of a novel Nudix hydrolase family whose members fold as asymmetric homodimers and act on bacterial cell wall precursors as well as RNA.…”

Section: Introductionmentioning

confidence: 99%

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A UDP-X Diphosphatase from Streptococcus pneumoniae Hydrolyzes Precursors of Peptidoglycan Biosynthesis

Duong‐Ly¹,

Woo²,

Dunn³

et al. 2013

PLoS ONE

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The gene for a Nudix enzyme (SP_1669) was found to code for a UDP-X diphosphatase. The SP_1669 gene is localized among genes encoding proteins that participate in cell division in Streptococcus pneumoniae. One of these genes, MurF, encodes an enzyme that catalyzes the last step of the Mur pathway of peptidoglycan biosynthesis. Mur pathway substrates are all derived from UDP-glucosamine and all are potential Nudix substrates. We showed that UDP-X diphosphatase can hydrolyze the Mur pathway substrates UDP-N-acetylmuramic acid and UDP-N-acetylmuramoyl-L-alanine. The 1.39 Å resolution crystal structure of this enzyme shows that it folds as an asymmetric homodimer with two distinct active sites, each containing elements of the conserved Nudix box sequence. In addition to its Nudix catalytic activity, the enzyme has a 3′5′ RNA exonuclease activity. We propose that the structural asymmetry in UDP-X diphosphatase facilitates the recognition of these two distinct classes of substrates, Nudix substrates and RNA. UDP-X diphosphatase is a prototype of a new family of Nudix enzymes with unique structural characteristics: two monomers, each consisting of an N-terminal helix bundle domain and a C-terminal Nudix domain, form an asymmetric dimer with two distinct active sites. These enzymes function to hydrolyze bacterial cell wall precursors and degrade RNA.

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“…These results indicate that UDP-Xase degrades RNA in the 3′ 5′ direction. This activity is similar to that of the B. cereus CDP-Chase [5].…”

Section: Resultssupporting

confidence: 78%

Section: Resultsmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A UDP-X Diphosphatase from Streptococcus pneumoniae Hydrolyzes Precursors of Peptidoglycan Biosynthesis

Duong‐Ly¹,

Woo²,

Dunn³

et al. 2013

PLoS ONE

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Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family

Nguyen

Park

et al. 2016

Proteins

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The nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74‐compound library of known Nudix enzyme substrates. We found substrates for four enzymes with k cat/K m values >10,000 M−1 s−1: Q92EH0_LISIN of Listeria innocua serovar 6a against ADP‐ribose, Q5LBB1_BACFN of Bacillus fragilis against 5‐Me‐CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8‐oxo‐dATP and 3'‐dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty‐two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported k cat/K m values exhibited against these canonical substrates are well under 105 M−1 s−1. By contrast, several Nudix enzymes show much larger k cat/K m values (in the range of 105 to >107 M−1 s−1) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810–1822. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

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The evolution of function within the Nudix homology clan

Srouji

Park

et al. 2017

Proteins

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The Nudix homology clan encompasses over 80,000 protein domains from all three domains of life, defined by homology to each other. Proteins with a domain from this clan fall into four general functional classes: pyrophosphohydrolases, isopentenyl diphosphate isomerases (IDIs), adenine/guanine mismatch-specific adenine glycosylases (A/G-specific adenine glycosylases), and non-enzymatic activities such as protein/protein interaction and transcriptional regulation. The largest group, pyrophosphohydrolases, encompasses more than 100 distinct hydrolase specificities. To understand the evolution of this vast number of activities, we assembled and analyzed experimental and structural data for 205 Nudix proteins collected from the literature. We corrected erroneous functions or provided more appropriate descriptions for 53 annotations described in the Gene Ontology Annotation database in this family, and propose 275 new experimentally-based annotations. We manually constructed a structure-guided sequence alignment of 78 Nudix proteins. Using the structural alignment as a seed, we then made an alignment of 347 “select” Nudix homology domains, curated from structurally determined, functionally characterized, or phylogenetically important Nudix domains. Based on our review of Nudix pyrophosphohydrolase structures and specificities, we further analyzed a loop region downstream of the Nudix hydrolase motif previously shown to contact the substrate molecule and possess known functional motifs. This loop region provides a potential structural basis for the functional radiation and evolution of substrate specificity within the hydrolase family. Finally, phylogenetic analyses of the 347 select protein domains and of the complete Nudix homology clan revealed general monophyly with regard to function and a few instances of probable homoplasy.

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

Cited by 10 publications

References 48 publications

A UDP-X Diphosphatase from Streptococcus pneumoniae Hydrolyzes Precursors of Peptidoglycan Biosynthesis

A UDP-X Diphosphatase from Streptococcus pneumoniae Hydrolyzes Precursors of Peptidoglycan Biosynthesis

Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family

The evolution of function within the Nudix homology clan

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