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Yokoyama, Shigeyuki; Harada, Hiroshi; Kigawa, T.; Yabuki, Takashi; Shirouzu, Mikako; Terada, Takaho; Ito, Yutaka; Matsuo, Yo; Kuroda, Yutaka; Nishimura, Y.; Kyōgoku, Yoshimasa; Miki, Kunio; Masui, Ryoji; Kuramitsu, Seiki

doi:10.1038/80712

Cited by 338 publications

(109 citation statements)

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“…Genome analysis of T. thermophilus HB8 suggested the absence of an ADPR-transferase like protein and the presence of an ADPR-glycohydrolase like protein, involved in ADPR transfer in mono-ADP-ribosylation (8,9). It implies that the possibility that ADPR molecules are constantly produced by mono-ADP-ribosylation is low.…”

Section: Discussionmentioning

confidence: 99%

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Structural Insights into the Thermus thermophilus ADP-ribose Pyrophosphatase Mechanism via Crystal Structures with the Bound Substrate and Metal

Yoshiba

Ooga

Nakagawa

et al. 2004

Journal of Biological Chemistry

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ADP-ribose pyrophosphatase (ADPRase) catalyzes the divalent metal ion-dependent hydrolysis of ADP-ribose to ribose 5 -phosphate and AMP. This enzyme plays a key role in regulating the intracellular ADP-ribose levels, and prevents nonenzymatic ADP-ribosylation. To elucidate the pyrophosphatase hydrolysis mechanism employed by this enzyme, structural changes occurring on binding of substrate, metal and product were investigated using crystal structures of ADPRase from an extreme thermophile, Thermus thermophilus HB8. Seven structures were determined, including that of the free enzyme, the Zn 2؉ -bound enzyme, the binary complex with ADP-ribose, the ternary complexes with ADPribose and Zn 2؉ or Gd 3؉ , and the product complexes with AMP and Mg 2؉ or with ribose 5 -phosphate and Zn 2؉ . The structural and functional studies suggested that the ADP-ribose hydrolysis pathway consists of four reaction states: bound with metal (I), metal and substrate (II), metal and substrate in the transition state (III), and products (IV). In reaction state II, Glu-82 and Glu-70 abstract a proton from a water molecule. This water molecule is situated at an ideal position to carry out nucleophilic attack on the adenosyl phosphate, as it is 3.6 Å away from the target phosphorus and almost in line with the scissile bond.Nudix pyrophosphatases are widely distributed in nature and share a highly conserved amino acid sequence motif called the "Nudix motif " (GX 5 EX 7 REUXEEXGU, where U is one of the bulky hydrophobic amino acids I, L, or V), which adopts a unique loop-helix-loop structure (1). Enzymes in this family catalyze the hydrolysis of nucleoside diphosphates, linked to another moiety x. Their postulated role is to control the cellular concentration of toxic nucleoside diphosphate derivatives or physiological metabolites, accumulation of which could be harmful (1). ADP-ribose (ADPR) 1 is one such diphosphate derivative, which is produced enzymatically as part of the turnover of NAD ϩ , cyclic ADPR, ADP-ribosylated proteins, and poly-ADP-ribosylated proteins. Although certain proteins are posttranslationally modified by ADPR, high intracellular levels of ADPR could result in nonenzymatic ADP-ribosylation. This is a deleterious process that inactivates enzymes and could interfere with the recognition of enzymatic ADP-ribosylation (2). ADPR pyrophosphatases (ADPRases) catalyze the hydrolysis of ADPR to AMP and ribose 5Ј-phosphate to prevent ADPR accumulation.ADPRase activity has been detected in all three kingdoms (3-7), but the specificity for ADPR over other substrates and the selectivity of metal ions required for activity vary between species. The mechanism underlying the different substrate specificity and the metal dependence is unknown at both the structural and functional levels. Elucidation of these properties requires the study of ADPRases from numerous sources.In this article, we investigated the catalytic mechanism of ADPRase from an extreme thermophile, Thermus thermophilus HB8 (TtADPRase). In general, proteins isolated fr...

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Section: Discussionmentioning

confidence: 99%

“…In general, proteins isolated from T. thermophilus are heat-stable and suitable for physicochemical studies, including x-ray crystallography (8,9). TtADPRase catalyzes the hydrolysis of ADPR to AMP and ribose 5Ј-phosphate in the presence of Mg 2ϩ and Zn 2ϩ ions.…”

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confidence: 99%

Structural Insights into the Thermus thermophilus ADP-ribose Pyrophosphatase Mechanism via Crystal Structures with the Bound Substrate and Metal

Yoshiba

Ooga

Nakagawa

et al. 2004

Journal of Biological Chemistry

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“…Crystallization and Data Collection-The purified protein was supplied by the RIKEN Structural Genomics Initiative (11). Crystallization of the native and complex of tAsS was carried out at 293 K by the hanging-drop vapor-diffusion method (12), using 30 mg/ml protein solution and 2.4 M ammonium sulfate, 30% (v/v) glycerol, 100 mM TrisHCl, pH 8.5, as the reservoir solution.…”

Section: Methodsmentioning

confidence: 99%

Crystal Structure of Argininosuccinate Synthetase fromThermus thermophilus HB8

Goto

Nakajima

Hirotsu

2002

Journal of Biological Chemistry

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Argininosuccinate synthetase catalyzes the ATP-dependent condensation of a citrulline with an aspartate to give argininosuccinate. The three-dimensional structures of the enzyme from Thermus thermophilus HB8 in its free form, complexed with intact ATP, and complexed with an ATP analogue (adenylyl imidodiphosphate) and substrate analogues (arginine and succinate) have been determined at 2.3-, 2.3-, and 1.95-Å resolution, respectively. The structure is essentially the same as that of the Escherichia coli argininosuccinate synthetase. The small domain has the same fold as that of a new family of "N-type" ATP pyrophosphatases with the P-loop specific for the pyrophosphate of ATP. However, the enzyme shows the P-loop specific for the ␥-phosphate of ATP. The structure of the complex form is quite similar to that of the native one, indicating that no conformational change occurs upon the binding of ATP and the substrate analogues. ATP and the substrate analogues are bound to the active site with their reaction sites close to one another and located in a geometrical orientation favorable to the catalytic action. The reaction mechanism so far proposed seems to be consistent with the locations of ATP and the substrate analogues. The reaction may proceed without the large conformational change of the enzyme proposed for the catalytic process.Argininosuccinate synthetase (AsS) 1 catalyzes the second step of the urea cycle where a citrulline, the product of the first step catalyzed by ornithine transcarbamoylase, reacts with an aspartate to give an argininosuccinate, which is cleaved into an arginine and a fumarate by argininosuccinase. AsS reversibly catalyzes the adenosine triphosphate (ATP)-dependent condensation of a citrulline with an aspartate (Scheme 1).The ureido oxygen atom of a citrulline attacks the ␣-P atom of the triphosphate in ATP to produce a citrullyl-AMP intermediate and a pyrophosphate as a leaving group. The AMP moiety of the activated citrullyl-AMP intermediate is displaced by an aspartate, thus producing an argininosuccinate (1, 2). Support for the existence of the citrullyl-AMP intermediate comes from experiments using 18 O-labeled citrulline (Scheme 1). Based on the structure of the ATP pyrophosphatase domain of the GMP synthetase (3) and the modified version of the P-loop of H-Ser-Gly-Gly-X-Asp-Ser/Thr-Ser/Thr (where H is any hydrophobic amino acid and X is any one) observed in GMP synthetase, NAD ϩ synthetase, asparagine synthetase, and AsS (4), these four distinct enzymes, which show ATP pyrophosphate activity, were proposed to have a common fold specific for nucleotide binding and belong to a new family of "N-type" ATP pyrophosphatases (3, 4). NAD ϩ synthetase (5, 6) and the Cterminal domain of asparagine synthetase (7) have been proved to be folded into an open ␣/␤ structure with a topology similar to that of the ATP pyrophosphate domain of GMP synthetase. In the structures of the GMP and NAD ϩ synthetases (3, 8), the P-loop specifically interacts with the ␤-and ␥-phosphates of ATP.AsS from The...

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“…Such use of MS can facilitate structure determination by X-ray diffraction or NMR (Cohen & Chait, 2001). MS is being used for quality assurance by structural genomics/ proteomics consortia in the USA (Pantazatos et al, 2004;Jeon et al, 2005), Japan (Yokoyama et al, 2000) and Europe Potier et al, 2003;Nettleship et al, 2005 Gervais et al, 2004). The method has been routinely used by some SPINE partners in cases where the structure determination of the full-length protein has failed.…”

Section: Introductionmentioning

confidence: 99%

The impact of protein characterization in structural proteomics

Geerlof

Brown

Coutard

et al. 2006

Acta Crystallogr D Biol Cryst

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Protein characterization plays a role in two key aspects of structural proteomics. The first is the quality assessment of the produced protein preparations. Obtaining well diffracting crystals is one of the major bottlenecks in the structuredetermination pipeline. Often, this is caused by the poor quality of the protein preparation used for crystallization trials. Hence, it is essential to perform an extensive quality assessment of the protein preparations prior to crystallization and to use the results in the evaluation of the process. Here, a protein-production and crystallization strategy is proposed with threshold values for protein purity (95%) and monodispersity (85%) below which a further optimization of the protein-production process is strongly recommended. The second aspect is the determination of protein characteristics such as domains, oligomeric state, post-translational modifications and protein-protein and protein-ligand interactions. In this paper, applications and new developments of proteincharacterization methods using MS, fluorescence spectroscopy, static light scattering, analytical ultracentrifugation and small-angle X-ray scattering within the EC Structural Proteomics in Europe contract are described. Examples of the application of the various methods are given.

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Cited by 338 publications

References 10 publications

Structural Insights into the Thermus thermophilus ADP-ribose Pyrophosphatase Mechanism via Crystal Structures with the Bound Substrate and Metal

Structural Insights into the Thermus thermophilus ADP-ribose Pyrophosphatase Mechanism via Crystal Structures with the Bound Substrate and Metal

Crystal Structure of Argininosuccinate Synthetase fromThermus thermophilus HB8

The impact of protein characterization in structural proteomics

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