Purification, characterization and crystallization of thermostable anthranilate phosphoribosyltransferase from <i>Sulfolobus solfataricus</i>

Ivens, Andreas; Mayans, Olga; Szadkowski, Halina; Wilmanns, Matthias; Kirschner, Kasper

doi:10.1046/j.1432-1327.2001.02102.x

Cited by 20 publications

(32 citation statements)

References 26 publications

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“…Crystal Structure Elucidation-ssAnPRT was overexpressed, purified, and crystallized as reported (12). Substrate complexation was in the crystalline state using soaking at 4°C, where crystals of the apoenzyme were immersed in cryo-solutions containing mother liquor supplemented with 22% (v/v) glycerol (as cryo-protectant) and ligands.…”

Section: Methodsmentioning

confidence: 99%

“…The turnover number k cat was obtained by dividing the maximum catalytic rate by the total concentration of active sites. (12). The crystal form employed in this study contained two AnPRT dimers in its asymmetric unit.…”

Section: Methodsmentioning

confidence: 99%

“…Samples were kept at 4°C throughout the irradiation experiments to prevent catalysis (12). The overall parameters of free and liganded ssAnPRT solutions are given in Table 2 and scattering patterns are shown in Fig.…”

Section: Crystalmentioning

confidence: 99%

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Structural and Mutational Analysis of Substrate Complexation by Anthranilate Phosphoribosyltransferase from Sulfolobus solfataricus

Marino

Deuss

Svergun

et al. 2006

Journal of Biological Chemistry

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The metabolic synthesis and degradation of essential nucleotide compounds are primarily carried out by phosphoribosyltransferases (PRT) and nucleoside phosphorylases (NP), respectively. Despite the resemblance of their reactions, five classes of PRTs and NPs exist, where anthranilate PRT (AnPRT) constitutes the only evolutionary link between synthesis and degradation processes. We have characterized the active site of dimeric AnPRT from Sulfolobus solfataricus by elucidating crystal structures of the wild-type enzyme complexed to its two natural substrates anthranilate and 5-phosphoribosyl-1-pyrophosphate/Mg 2؉ . These bind into two different domains within each protomer and are brought together during catalysis by rotational domain motions as shown by small angle x-ray scattering data. Steady-state kinetics of mutated AnPRT variants address the role of active site residues in binding and catalysis. Results allow the comparative analysis of PRT and pyrimidine NP families and expose related structural motifs involved in nucleotide/ nucleoside recognition by these enzyme families.Nucleotide compounds are central to the production of genetic material, the amino acids histidine and tryptophan, and cofactors such as NAD. Their metabolism is largely based on the reversible transfer of a phosphoribosyl group to aromatic bases. Although forward and reverse ribosylation processes share a close resemblance (Fig. 1), they are carried out by different enzymes and in the context of distinct metabolic pathways. Synthesis reactions are performed by phosphoribosyltransferases (PRT). 3 These use PRPP as universal phosphoribosyl donor. These reactions, which are dependent on metal ions, involve the displacement of the 1-pyrophosphate group of PRPP and formation of a N-1Ј-glycosidic bond to a nitrogenated base specific for each PRT. Conversely, the release of aromatic bases from nucleotides involves first the production of a nucleoside intermediate by nucleotidases or phosphatases, followed by the cleavage of the glycosidic bond by either nucleoside phosphorylases (NP) or nucleoside hydrolases. NP enzymes are key to nucleotide salvage both in prokaryotes and in eukaryotes. They catalyze the reversible phosphorolysis of the glycosidic bond of nucleosides to yield free bases and ribose-1-phosphate.Despite the chemical resemblance of the compounds intervening in synthesis and degradation reactions by PRTs and NPs ( Fig. 1), respectively, the enzymes involved in these catalyses lack structural similarity. Three diverse PRT classes that act on aromatic bases are known to date, as follows: (i) those with a canonical fold classified as type PRT-I, which includes most of the PRTs for which an atomic structure is available (1); (ii) type PRT-II comprising quinolate PRT (2-3) and nicotinate PRT (4); and (iii) anthranilate PRT (AnPRT) (5-7) (Fig. 1A). NPs are also classified in two groups with distinct folds as follows: (i) NP-I, which act primarily on purine nucleosides but also accept the pyrimidine uridine, and (ii) NP-II, which degrade t...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Structural and Mutational Analysis of Substrate Complexation by Anthranilate Phosphoribosyltransferase from Sulfolobus solfataricus

Marino

Deuss

Svergun

et al. 2006

Journal of Biological Chemistry

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“…Binding of PRPP and anthranilate is mediated in a cleft of the ␣/␤-domain. The binding of PRPP is promoted by two Mg 2ϩ or Mn 2ϩ , which accounts for a requirement of Mg 2ϩ for activity (288). The binding of PRPP has been well mapped in the structures of S. solfataricus and M. tuberculosis anthranilate phosphoribosyltransferase.…”

Section: Reactions At the Anomeric Carbon Of Prppmentioning

confidence: 99%

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

Hove‐Jensen

Andersen

Kilstrup

et al. 2017

Microbiol Mol Biol Rev

168

187

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SUMMARY Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species.

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“…From chorismate to tyrosine and phenylalanine, chorismate mutase (CM) from M. jannaschii, prephenate dehydratase (PDT) from Halobacterium vallismortis, and aromatic aminotransferases (AroAT) from Methanococcus aeolicus and Pyrococcus furiosus have been characterized (27,34,62,69). Most of the enzymes for the biosynthesis of tryptophan from chorismate have been characterized in archaea, including some Methanococcus species (14,24,29,38,48,55,61).…”

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confidence: 99%

Two Biosynthetic Pathways for Aromatic Amino Acids in the Archaeon Methanococcus maripaludis

Porat¹,

Waters²,

Teng

et al. 2004

J Bacteriol

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Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon. Aromatic amino acids (AroAAs) are biosynthesized in this autotroph either by the de novo pathway, with chorismate as an intermediate, or by the incorporation of exogenous aryl acids via indolepyruvate oxidoreductase (IOR). In order to evaluate the roles of these pathways, the gene that encodes the third step in the de novo pathway, 3-dehydroquinate dehydratase (DHQ), was deleted. This mutant required all three AroAAs for growth, and no DHQ activity was detectible in cell extracts, compared to 6.0 ؎ 0.2 mU mg ؊1 in the wild-type extract. The growth requirement for the AroAAs could be fulfilled by the corresponding aryl acids phenylacetate, indoleacetate, and p-hydroxyphenylacetate. The specific incorporation of phenylacetate into phenylalanine by the IOR pathway was demonstrated in vivo by labeling with [1-13 C]phenylacetate. M. maripaludis has two IOR homologs. A deletion mutant for one of these homologs contained 76, 74, and 42% lower activity for phenylpyruvate, p-hydoxyphenylpyruvate, and indolepyruvate oxidation, respectively, than the wild type. Growth of this mutant in minimal medium was inhibited by the aryl acids, but the AroAAs partially restored growth. Genetic complementation of the IOR mutant also restored much of the wild-type phenotype. Thus, aryl acids appear to regulate the expression or activity of the de novo pathway. The aryl acids did not significantly inhibit the activity of the biosynthetic enzymes chorismate mutase, prephenate dehydratase, and prephenate dehydrogenase in cell extracts, so the inhibition of growth was probably not due to an effect on these enzymes.

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Purification, characterization and crystallization of thermostable anthranilate phosphoribosyltransferase from Sulfolobus solfataricus

Cited by 20 publications

References 26 publications

Structural and Mutational Analysis of Substrate Complexation by Anthranilate Phosphoribosyltransferase from Sulfolobus solfataricus

Structural and Mutational Analysis of Substrate Complexation by Anthranilate Phosphoribosyltransferase from Sulfolobus solfataricus

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

Two Biosynthetic Pathways for Aromatic Amino Acids in the Archaeon Methanococcus maripaludis

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