Structural studies of the Nudix GDP-mannose hydrolase from E. coli reveals a new motif for mannose recognition

The Nudix hydrolase superfamily, characterized by the presence of the signature sequence GX(5)EX(7)REUXEEXGU (where U is I, L, or V), is a well-studied family in which relations have been established between primary sequence and substrate specificity for many members. For example, enzymes that hydrolyze the diphosphate linkage of ADP-ribose are characterized by having a proline 15 amino acids C-terminal of the Nudix signature sequence. GDPMK is a Nudix enzyme that conserves this characteristic proline but uses GDP-mannose as the preferred substrate. By investigating the structure of the GDPMK alone, bound to magnesium, and bound to substrate, the structural basis for this divergent substrate specificity and a new rule was identified by which ADP-ribose pyrophosphatases can be distinguished from purine-DP-mannose pyrophosphatases from primary sequence alone. Kinetic and mutagenesis studies showed that GDPMK hydrolysis does not rely on a single glutamate as the catalytic base. Instead, catalysis is dependent on residues that coordinate the magnesium ions and residues that position the substrate properly for catalysis. GDPMK was thought to play a role in biofilm formation because of its upregulation in response to RcsC signaling; however, GDPMK knockout strains show no defect in their capacity of forming biofilms.     

Structure and mechanism of MT-ADPRase,a nudix hydrolase from Mycobacterium tuberculosis

Nudix hydrolases are a family of proteins that contain the characteristic sequence GX(5)EX(7)REUXEEXG(I/L/V), the Nudix box. They catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives such as ADP-ribose, Ap(n)A (3 相似文献   

Structure and mechanism of GDP-mannose glycosyl hydrolase, a Nudix enzyme that cleaves at carbon instead of phosphorus

GDP-mannose glycosyl hydrolase (GDPMH) catalyzes the hydrolysis of GDP-mannose and GDP-glucose to GDP and sugar by substitution with inversion at C1 of the sugar. The enzyme has a modified Nudix motif and requires one divalent cation for activity. The 1.3 A X-ray structure of the GDPMH-Mg(2+)-GDP complex, together with kinetic, mutational, and NMR data, suggests a mechanism for the GDPMH reaction. Several residues and the divalent cation strongly promote the departure of the GDP leaving group, supporting a dissociative mechanism. Comparison of the GDPMH structure with that of a typical Nudix hydrolase suggests how sequence changes result in the switch of catalytic activity from P-O bond cleavage to C-O bond cleavage. Changes in the Nudix motif result in loss of binding of at least one Mg(2+) ion, and shortening of a loop by 6 residues shifts the catalytic base by approximately 10 A.     

The crystal structure of diadenosine tetraphosphate hydrolase from Caenorhabditis elegans in free and binary complex forms

The crystal structure of C. elegans Ap(4)A hydrolase has been determined for the free enzyme and a binary complex at 2.0 A and 1.8 A, respectively. Ap(4)A hydrolase has a key role in regulating the intracellular Ap(4)A levels and hence potentially the cellular response to metabolic stress and/or differentiation and apoptosis via the Ap(3)A/Ap(4)A ratio. The structures reveal that the enzyme has the mixed alpha/beta fold of the Nudix family and also show how the enzyme binds and locates its substrate with respect to the catalytic machinery of the Nudix motif. These results suggest how the enzyme can catalyze the hydrolysis of a range of related dinucleoside tetraphosphate, but not triphosphate, compounds through precise orientation of key elements of the substrate.     

The crystal structure and mutational analysis of human NUDT9

Human ADP-ribose pyrophosphatase NUDT9 belongs to a superfamily of Nudix hydrolases that catabolize potentially toxic compounds in the cell. The enzyme hydrolyzes ADP-ribose (ADPR) to AMP and ribose 5'-phosphate. NUDT9 shares 39% sequence identity with the C-terminal cytoplasmic domain of the ADPR-gated calcium channel TRPM2, which exhibits low but specific enzyme activity. We determined crystal structures of NUDT9 in the presence and in the absence of the reaction product ribose 5'-phosphate. On the basis of these structures and comparison with a bacterial homologue, a model of the substrate complex was built. The structure and activity of a double point mutant (R(229)E(230)F(231) to R(229)I(230)L(231)), which mimics the Nudix signature of the ion channel domain, was determined. Finally, the activities of a pair of additional mutated constructs were compared to the wild-type enzyme. The first corresponds to a minimal Nudix domain missing an N-terminal domain and C-terminal tail; the second disrupts two potential general bases in the active site. NUDT9 contains an N-terminal domain with a novel fold and a catalytic C-terminal Nudix domain. Unlike its closest functional homologue (homodimeric Escherichia coli ADPRase), it is active as a monomer, and the substrate is bound in a cleft between the domains. The structure of the RIL mutant provides structural basis for the reduced activity of the TRPM2 ion channel. The conformation and binding interactions of ADPR substrate are predicted to differ from those observed for E.coli ADPRase; mutation of structurally aligned acidic residues in their active sites produce significantly different effects on catalytic efficiency, indicating that their reaction pathways and mechanisms may have diverged.     

Molecular mechanism of the Thermus thermophilus ADP-ribose pyrophosphatase from mutational and kinetic studies

ADP-ribose pyrophosphatase (ADPRase), a member of the nudix protein family, catalyzes the hydrolysis of ADP-ribose to AMP and ribose 5'-phosphate. We have determined the crystal structure of ADPRase from Thermus thermophilus HB8 (TtADPRase). We performed kinetic analysis of mutants of TtADPRase to elucidate the substrate recognition and the catalytic mechanism. Our results suggest that interactions responsible for the substrate recognition are located at the terminal moieties of the substrate. The adenine moiety is recognized by Ile-19 and the main chain carbonyl group of Glu-29 and/or Gly-104. The terminal ribose moiety is recognized by the sum of some weak interactions with multiple residues that are close in space. Glu-82 and Glu-86, conserved in the nudix motif, were previously shown to be essential for catalysis. Mutation of these residues shows that the dependence of kcat on pH is almost the same as that of the wild-type enzyme. Results suggest that Glu-82 and Glu-86 are essential for catalysis but unlikely to act as a catalytic base. In the crystal structure, each acidic residue coordinates with a metal ion. Furthermore, a water molecule coordinates between these two metals. Our results suggest a two-metal ion mechanism for the catalysis of ADPRase in which a water molecule is activated to act as a nucleophile by the cations coordinated by Glu-82 and Glu-86. Arg-54, Glu-70, Arg-81, and Glu-85 are predicted to support this nucleophilic attack on the alpha-phosphate of the substrate. Interestingly, ADPRase displays differences in the substrate recognition and the catalytic mechanism from the models proposed for other nudix proteins. Our results highlight the diversity within the nudix protein family in terms of substrate recognition and catalysis.     

Molecular mechanism of ADP-ribose hydrolysis by human NUDT5 from structural and kinetic studies

Human NUDT5 (hNUDT5) is an ADP-ribose (ADPR) pyrophosphatase (ADPRase) that plays important roles in controlling the intracellular levels of ADPR and preventing non-enzymatic ADP-ribosylation of proteins by hydrolyzing ADPR to AMP and ribose 5′-phosphate. We report the crystal structure of hNUDT5 in complex with a non-hydrolyzable ADPR analogue, α,β-methyleneadenosine diphosphoribose, and three Mg^2 + ions representing the transition state of the enzyme during catalysis. Analysis of this structure and comparison with previously reported hNUDT5 structures identify key residues involved in substrate binding and catalysis. In the transition-state structure, three metal ions are bound at the active site and are coordinated by surrounding residues and water molecules. A conserved water molecule is at an ideal position for nucleophilic attack on the α-phosphate of ADPR. The side chain of Glu166 on loop L9 changes its conformation to interact with the conserved water molecule compared with that in the substrate-bound structure and appears to function as a catalytic base. Mutagenesis and kinetic studies show that Trp28 and Trp46 are important for the substrate binding; Arg51 is involved in both the substrate binding and the catalysis; and Glu112 and Glu116 of the Nudix motif, Glu166 on loop L9, and Arg111 are critical for the catalysis. The structural and biochemical data together reveal the molecular basis of the catalytic mechanism of ADPR hydrolysis by hNUDT5. Specifically, Glu166 functions as a catalytic base to deprotonate a conserved water molecule that acts as a nucleophile to attack the α-phosphate of ADPR, and three Mg^2 + ions are involved in the activation of the nucleophile and the binding of the substrate. Structural comparison of different ADPRases also suggests that most dimeric ADPRases may share a similar catalytic mechanism of ADPR hydrolysis.     

Structural basis for different substrate specificities of two ADP-ribose pyrophosphatases from Thermus thermophilus HB8

ADP-ribose (ADPR) is one of the main substrates of Nudix proteins. Among the eight Nudix proteins of Thermus thermophilus HB8, we previously determined the crystal structure of Ndx4, an ADPR pyrophosphatase (ADPRase). In this study we show that Ndx2 of T. thermophilus also preferentially hydrolyzes ADPR and flavin adenine dinucleotide and have determined its crystal structure. We have determined the structures of Ndx2 alone and in complex with Mg2+, with Mg2+ and AMP, and with Mg2+ and a nonhydrolyzable ADPR analogue. Although Ndx2 recognizes the AMP moiety in a manner similar to those for other ADPRases, it recognizes the terminal ribose in a distinct manner. The residues responsible for the recognition of the substrate in Ndx2 are not conserved among ADPRases. This may reflect the diversity in substrate specificity among ADPRases. Based on these results, we propose the classification of ADPRases into two types: ADPRase-I enzymes, which exhibit high specificity for ADPR; and ADPRase-II enzymes, which exhibit low specificity for ADPR. In the active site of the ternary complexes, three Mg2+ ions are coordinated to the side chains of conserved glutamate residues and water molecules. Substitution of Glu90 and Glu94 with glutamine suggests that these residues are essential for catalysis. These results suggest that ADPRase-I and ADPRase-II enzymes have nearly identical catalytic mechanisms but different mechanisms of substrate recognition.     

Site-directed mutagenesis of diphosphoinositol polyphosphate phosphohydrolase, a dual specificity NUDT enzyme that attacks diadenosine polyphosphates and diphosphoinositol polyphosphates

Diphosphoinositol polyphosphate phosphohydrolase (DIPP) hydrolyzes diadenosine 5',5"'-P(1),P(6)-hexaphosphate (Ap(6)A), a Nudix (nucleoside diphosphate attached-moiety "x") substrate, and two non-Nudix compounds: diphosphoinositol pentakisphosphate (PP-InsP(5)) and bis-diphosphoinositol tetrakisphosphate ((PP)(2)-InsP(4)). Guided by multiple sequence alignments, we used site-directed mutagenesis to obtain new information concerning catalytically essential amino acid residues in DIPP. Mutagenesis of either of two conserved glutamate residues (Glu(66) and Glu(70)) within the Nudt (Nudix-type) catalytic motif impaired hydrolysis of Ap(6)A, PP-InsP(5), and (PP)(2)-InsP(4) >95%; thus, all three substrates are hydrolyzed at the same active site. Two Gly-rich domains (glycine-rich regions 1 and 2 (GR1 and GR2)) flank the Nudt motif with potential sites for cation coordination and substrate binding. GR1 comprises a GGG tripeptide, while GR2 is identified as a new functional motif (GX(2)GX(6)G) that is conserved in yeast homologues of DIPP. Mutagenesis of any of these Gly residues in GR1 and GR2 reduced catalytic activity toward all three substrates by up to 95%. More distal to the Nudt motif, H91L and F84Y mutations substantially decreased the rate of Ap(6)A and (PP)(2)-InsP(4) metabolism (by 71 and 96%), yet PP-InsP(5) hydrolysis was only mildly reduced (by 30%); these results indicate substrate-specific roles for His(91) and Phe(84). This new information helps define DIPP's structural, functional, and evolutionary relationships to Nudix hydrolases.     

Identification and characterization of NuhA, a novel Nudix hydrolase specific for ADP-ribose in the cyanobacterium Synechococcus sp. PCC 7002

We cloned the gene for a novel Nudix hydrolase in the cyanobacterium Synechococcus sp. PCC 7002 and termed it nuhA. The deduced amino acid sequence of NuhA included the Nudix motif, GX(5)EX(7)RELXEEXGV, which is common to Nudix hydrolases, and in addition, a proline at the 15th amino acid from the C-terminus of the Nudix motif, which is characteristic of the subfamily of ADP-ribose pyrophosphatases. The recombinant NuhA with a hexahistidine tag was overexpressed in Escherichia coli and purified. The recombinant NuhA hydrolyzed ADP-ribose specifically among various nucleoside diphosphate derivatives. The hydrolytic activity for ADP-ribose required Mg(2+) and was optimal at pH 9.5. The V(max) and K(m) values of hydrolysis were 23.6 units mg(-1) and 0.094 mM, respectively. NuhA contained an uncharacterized domain in the C-terminal region, termed Pfam-B-3116, which is conserved in several hypothetical proteins. The mutated NuhA deficient in the Pfam-B-3116 domain failed to form the hexamers that are characteristic of NuhA, and exhibited a significantly higher K(m) value for ADP-ribose, suggesting that the Pfam-B-3116 domain might be responsible for oligomerization of NuhA and full binding affinity for ADP-ribose. These unique features suggest that NuhA is a novel type of ADP-ribose pyrophosphatase.     

The Nudix hydrolase Ndx1 from Thermus thermophilus HB8 is a diadenosine hexaphosphate hydrolase with a novel activity

The ndx1 gene, which encodes a Nudix protein, was cloned from the extremely thermophilic bacterium Thermus thermophilus HB8. This gene encodes a 126-amino acid protein that includes the characteristic Nudix motif conserved among Nudix proteins. Ndx1 was overexpressed in Escherichia coli and purified. Ndx1 was stable up to 95 degrees C and at extreme pH. Size exclusion chromatography indicated that Ndx1 was monomeric in solution. Ndx1 specifically hydrolyzed (di)adenosine polyphosphates but not ATP or diadenosine triphosphate, and it always generated ATP as the product. Diadenosine hexaphosphate (Ap(6)A), the most preferred substrate, was hydrolyzed to produce two ATP molecules, which is a novel hydrolysis mode for Ap(6)A, with a K(m) of 1.4 microm and a k(cat) of 4.1 s(-1). These results indicate that Ndx1 is a (di)adenosine polyphosphate hydrolase. Ndx1 activity required the presence of the divalent cations Mn(2+), Mg(2+), Zn(2+), and Co(2+), whereas Ca(2+), Ni(2+), and Cu(2+) were not able to activate Ndx1. Fluoride ion inhibited Ndx1 activity via a non-competitive mechanism. Optimal activity for Ap(6)A was observed at around pH 8.0 and about 70 degrees C. We found two important residues with pK(a) values of 6.1 and 9.6 in the free enzyme and pK(a) values of 7.9 and 10.0 in the substrate-enzyme complex. Kinetic studies of proteins with amino acid substitutions suggested that Glu-46 and Glu-50 were conserved residues in the Nudix motif and were involved in catalysis. Trp-26 was likely involved in enzyme-substrate interactions based on fluorescence measurements. Based on these results, the mechanism of substrate recognition and catalysis are discussed.     

Mechanism of the Escherichia coli ADP-ribose pyrophosphatase,a Nudix hydrolase

Escherichia coli ADP-ribose (ADPR) pyrophosphatase (ADPRase), a Nudix enzyme, catalyzes the Mg(2+)-dependent hydrolysis of ADP-ribose to AMP and ribose 5-phosphate. ADPR hydrolysis experiments conducted in the presence of H(2)(18)O and analyzed by electrospray mass spectrometry showed that the ADPRase-catalyzed reaction takes place through nucleophilic attack at the adenosyl phosphate. The structure of ADPRase in complex with Mg(2+) and a nonhydrolyzable ADPR analogue, alpha,beta-methylene ADP-ribose, reveals an active site water molecule poised for nucleophilic attack on the adenosyl phosphate. This water molecule is activated by two magnesium ions, and its oxygen contacts the target phosphorus (P-O distance of 3.0 A) and forms an angle of 177 degrees with the scissile bond, suggesting an associative mechanism. A third Mg(2+) ion bridges the two phosphates and could stabilize the negative charge of the leaving group, ribose 5-phosphate. The structure of the ternary complex also shows that loop L9 moves fully 10 A from its position in the free enzyme, forming a tighter turn and bringing Glu 162 to its catalytic position. These observations indicate that as part of the catalytic mechanism, the ADPRase cycles between an open (free enzyme) and a closed (substrate-metal complex) conformation. This cycling may be important in preventing nonspecific hydrolysis of other nucleotides.     

The structure of Ap(4)A hydrolase complexed with ATP-MgF(x) reveals the basis of substrate binding

Ap(4)A hydrolases are Nudix enzymes that regulate intracellular dinucleoside polyphosphate concentrations, implicating them in a range of biological events, including heat shock and metabolic stress. We have demonstrated that ATP x MgF(x) can be used to mimic substrates in the binding site of Ap(4)A hydrolase from Lupinus angustifolius and that, unlike previous substrate analogs, it is in slow exchange with the enzyme. The three-dimensional structure of the enzyme complexed with ATP x MgF(x) was solved and shows significant conformational changes. The substrate binding site of L. angustifolius Ap(4)A hydrolase differs markedly from the two previously published Nudix enzymes, ADP-ribose pyrophosphatase and MutT, despite their common fold and the conservation of active site residues. The majority of residues involved in substrate binding are conserved in asymmetrical Ap(4)A hydrolases from pathogenic bacteria, but are absent in their human counterparts, suggesting that it might be possible to generate compounds that target bacterial, but not human, Ap(4)A hydrolases.     

Structures and mechanisms of Nudix hydrolases

Nudix hydrolases catalyze the hydrolysis of nucleoside diphosphates linked to other moieties, X, and contain the sequence motif or Nudix box, GX(5)EX(7)REUXEEXGU. The mechanisms of Nudix hydrolases are highly diverse in the position on the substrate at which nucleophilic substitution occurs, and in the number of required divalent cations. While most proceed by associative nucleophilic substitutions by water at specific internal phosphorus atoms of a diphosphate or polyphosphate chain, members of the GDP-mannose hydrolase sub-family catalyze dissociative nucleophilic substitutions, by water, at carbon. The site of substitution is likely determined by the positions of the general base and the entering water. The rate accelerations or catalytic powers of Nudix hydrolases range from 10(9)- to 10(12)-fold. The reactions are accelerated 10(3)-10(5)-fold by general base catalysis by a glutamate residue within, or beyond the Nudix box, or by a histidine beyond the Nudix box. Lewis acid catalysis, which contributes 10(3)-10(5)-fold to the rate acceleration, is provided by one, two, or three divalent cations. One divalent cation is coordinated by two or three conserved residues of the Nudix box, the initial glycine and one or two glutamate residues, together with a remote glutamate or glutamine ligand from beyond the Nudix box. Some Nudix enzymes require one (MutT) or two additional divalent cations (Ap(4)AP), to neutralize the charge of the polyphosphate chain, to help orient the attacking hydroxide or oxide nucleophile, and/or to facilitate the departure of the anionic leaving group. Additional catalysis (10-10(3)-fold) is provided by the cationic side chains of lysine and arginine residues and by H-bond donation by tyrosine residues, to orient the general base, or to promote the departure of the leaving group. The overall rate accelerations can be explained by both independent and cooperative effects of these catalytic components.     

Structural and functional diversity of Nudix fold

Nudix hydrolases catalyze the hydrolysis of nucleoside diphosphates linked to other moieties X, and contain the sequence motif or Nudix box, GX5EX7REUXEEXGU. The mechanisms of Nudix hydrolases are highly diverse in the position on the substrate. In this paper, we examined the sequences and structures of the MutT/Nudix superfamily. And two recent developed methods were employed for data analyses of the superfamily. One is QH method evaluating the similarities among structures for structural phylogeny. The other method is clustering analysis by using the CLANS program that could analysis thousands of sequences of the full dataset rather than the representative sequences of the superfamily. Finally, we proposed a more objective classification of the MutT/Nudix superfamily members based on detailed sequence and structure analyses.     

Crystal structures representing the Michaelis complex and the thiouronium reaction intermediate of Pseudomonas aeruginosa arginine deiminase

L-arginine deiminase (ADI) catalyzes the irreversible hydrolysis of L-arginine to citrulline and ammonia. In a previous report of the structure of apoADI from Pseudomonas aeruginosa, the four residues that form the catalytic motif were identified as Cys406, His278, Asp280, and Asp166. The function of Cys406 in nucleophilic catalysis has been demonstrated by transient kinetic studies. In this study, the structure of the C406A mutant in complex with L-arginine is reported to provide a snapshot of the enzyme.substrate complex. Through the comparison of the structures of apoenzyme and substrate-bound enzyme, a substrate-induced conformational transition, which might play an important role in activity regulation, was discovered. Furthermore, the position of the guanidinium group of the bound substrate relative to the side chains of His278, Asp280, and Asp166 indicated that these residues mediate multiple proton transfers. His278 and Asp280, which are positioned to activate the water nucleophile in the hydrolysis of the S-alkylthiouronium intermediate, were replaced with alanine to stabilize the intermediate for structure determination. The structures determined for the H278A and D280A mutants co-crystallized with L-arginine provide a snapshot of the S-alkylthiouronium adduct formed by attack of Cys406 on the guanidinium carbon of L-arginine followed by the elimination of ammonia. Asp280 and Asp166 engage in ionic interactions with the guanidinium group in the C406A ADI. L-arginine structure and might orient the reaction center and participate in proton transfer. Structure determination of D166A revealed the apoD166A ADI. The collection of structures is interpreted in the context of recent biochemical data to propose a model for ADI substrate recognition and catalysis.     

Structure of a coenzyme A pyrophosphatase from Deinococcus radiodurans: a member of the Nudix family

Gene Dr1184 from Deinococcus radiodurans codes for a Nudix enzyme (DR-CoAse) that hydrolyzes the pyrophosphate moiety of coenzyme A (CoA). Nudix enzymes with the same specificity have been found in yeast, humans, and mice. The three-dimensional structure of DR-CoAse, the first of a Nudix hydrolase with this specificity, reveals that this enzyme contains, in addition to the fold observed in other Nudix enzymes, insertions that are characteristic of a CoA-hydrolyzing Nudix subfamily. The structure of the complex of the enzyme with Mg(2+), its activating cation, reveals the position of the catalytic site. A helix, part of the N-terminal insertion, partially occludes the binding site and has to change its position to permit substrate binding. Comparison of the structure of DR-CoAse to those of other Nudix enzymes, together with the location in the structure of the sequence characteristic of CoAses, suggests a mode of binding of the substrate to the enzyme that is compatible with all available data.     

The 26 Nudix hydrolases of Bacillus cereus, a close relative of Bacillus anthracis

The genome of Bacillus cereus contains 26 Nudix hydrolase genes, second only to its closest relative, Bacillus anthracis which has 30. All 26 genes have been cloned, 25 have been expressed, and 21 produced soluble proteins suitable for analysis. Substrates for 16 of the enzymes were identified; these included ADP-ribose, diadenosine polyphosphates, sugar nucleotides, and deoxynucleoside triphosphates. One of the enzymes was a CDP-choline pyrophosphatase, the first Nudix hydrolase active on this substrate. Furthermore, as a result of this and previous work we have identified a new sub-family of the Nudix hydrolase superfamily recognizable by a specific amino acid motif outside of the Nudix box.