Characterization of the adenine nucleoside specific phosphorylase of Bacillus cereus

Sgarrella, Francesco; Frassetto, Luciano; Allegrini, Simone; Camici, Marcella; Carta, Mario; Fadda, Paolo; Tozzi, Maria Grazia; Ipata, Piero Luigi

doi:10.1016/j.bbagen.2007.07.004

Cited by 7 publications

(11 citation statements)

References 35 publications

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“…These analyses suggest that the negative cooperativity model of phosphate binding displayed by EcPNP cannot be applied for BcAdoP, as BcAdoP apparently presents only one conformational state. This hypothesis is supported by functional studies, which showed that BcAdoP obeys Michaelis–Menten kinetics [77].…”

Section: Resultsmentioning

confidence: 74%

Insights into Phosphate Cooperativity and Influence of Substrate Modifications on Binding and Catalysis of Hexameric Purine Nucleoside Phosphorylases

et al. 2012

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The hexameric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233) displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. In order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexameric BsPNP233. Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2′deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (2′deoxy)ribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 5′ hydroxyl group of adenosine and Arg43* side chain contributes for the ribosyl radical to adopt an unusual C3’-endo conformation. The structures with 6-chloroguanosine and 8-bromoguanosine pointed out that the Cl6 and Br8 substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens. Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by tubercidin and suggested that the acyclic nucleoside ganciclovir is a better inhibitor for hexameric PNPs than aciclovir. Furthermore, comparative structural analyses indicated that the replacement of Ser90 by a threonine in the B. cereus hexameric adenosine phosphorylase (Thr91) is responsible for the lack of negative cooperativity of phosphate binding in this enzyme.

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Section: Resultsmentioning

confidence: 74%

Insights into Phosphate Cooperativity and Influence of Substrate Modifications on Binding and Catalysis of Hexameric Purine Nucleoside Phosphorylases

et al. 2012

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“…However, because MTAP did not cleave methylAdo, only Ado-PNP could be responsible for the methyl-Ado cleavage observed in cell cultures. The K m app values for Ade (30). Our kinetic studies showed that Ado-PNP has a lower K m app for Ade than Ado, suggesting that in vitro, the formation of Ado from Ade is favored.…”

Section: Discussionmentioning

confidence: 92%

“…Similar to E. coli, B. subtilis and B. cereus also express two nucleoside phosphorylases, namely, inosine-guanosine phosphorylase and AdoP (16,29,30). Sgarrella et al described an Ado-specific phosphorylase (AdoP) in B. cereus and have shown that AdoP could cleave Ado and 2Ј-deoxyadenosine (30). Ino could also be cleaved by this enzyme with 1.5% of the relative activity of Ado, and no activity was observed with guanosine, xanthosine, or the pyrimidine nucleosides.…”

Section: Discussionmentioning

confidence: 99%

“…Although most bacterial purine nucleoside phosphorylases (PNPs) (EC 2.4.2.1) can cleave Ado to Ade, it is known that mycobacterial PNP does not accept Ado as a substrate (5,11,19). Some bacteria, such as Bacillus subtilis and Bacillus cereus, express an Ado phosphorylase (AdoP) that selectively cleaves Ado (16,29,30 Given that the literature indicates that a number of bacterial, parasitic, and mammalian enzymes can cleave Ado to Ade, we investigated Ado cleavage in cell extracts from M. smegmatis, a closely related, fast-growing model of M. tuberculosis. Ado cleavage activity was assayed in all the fractions eluting from our first purification column.…”

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confidence: 99%

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Identification and Characterization of Two Adenosine Phosphorylase Activities in Mycobacterium smegmatis

Buckoreelall

Wilson

Parker

2011

Journal of Bacteriology

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Purine nucleoside phosphorylase (PNP) is an important enzyme in purine metabolism and cleaves purine nucleosides to their respective bases. Mycobacterial PNP is specific for 6-oxopurines and cannot account for the adenosine (Ado) cleavage activity that has been detected in M. tuberculosis and M. smegmatis cultures. In the current work, two Ado cleavage activities were identified from M. smegmatis cell extracts. The first activity was biochemically determined to be a phosphorylase that could reversibly catalyze adenosine ؉ phosphate 7 adenine ؉ alpha-D-ribose-1-phosphate. Our purification scheme led to a 30-fold purification of this activity, with the removal of more than 99.9% of total protein. While Ado was the preferred substrate, inosine and guanosine were also cleaved, with 43% and 32% of the Ado activity, respectively. Our data suggest that M. smegmatis expresses two PNPs: a previously described trimeric PNP that can cleave inosine and guanosine only and a second, novel PNP (Ado-PNP) that can cleave Ado, inosine, and guanosine. Ado-PNP had an apparent K m (K m app ) of 98 ؎ 6 M (with Ado) and a native molecular mass of 125 ؎ 7 kDa. The second Ado cleavage activity was identified as 5-methylthioadenosine phosphorylase (MTAP) based on its biochemical properties and mass spectrometry analysis. Our study marks the first report of the existence of MTAP in any bacterium. Since human cells do not readily convert Ado to Ade, an understanding of the substrate preferences of these enzymes could lead to the identification of Ado analogs that could be selectively activated to toxic products in mycobacteria.

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“…Whereas trimeric PNPs are only active on 6-oxopurine nucleosides, hexameric PNPs generally accept both 6-oxo and 6-aminopurine nucleosides as substrates (Bzowska et al, 1990(Bzowska et al, , 2000; however, there are a few exceptions. For instance, hexameric PNP from Plasmodium falciparum does not cleave adenosine (Daddona et al, 1986), while hexameric B. cereus PNP is specific for adenosine (Sgarrella et al, 2007). Although the two classes of PNP do not share significant sequence similarity, they are structurally similar, having a protomeric fold consisting of a distorted -barrel flanked by -helices on each side.…”

Section: Introductionmentioning

confidence: 99%

Structure of grouper iridovirus purine nucleoside phosphorylase

Kang

Zhang

Allan

et al. 2010

Acta Crystallogr D Biol Cryst

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Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides to the corresponding free bases and ribose 1-phosphate. The crystal structure of grouper iridovirus PNP (givPNP), corresponding to the first PNP gene to be found in a virus, was determined at 2.4 Å resolution. The crystals belonged to space group R3, with unit-cell parameters a = 193.0, c = 105.6 Å , and contained four protomers per asymmetric unit. The overall structure of givPNP shows high similarity to mammalian PNPs, having an / structure with a nine-stranded mixed -barrel flanked by a total of nine -helices. The predicted phosphate-binding and ribose-binding sites are occupied by a phosphate ion and a Tris molecule, respectively. The geometrical arrangement and hydrogen-bonding patterns of the phosphate-binding site are similar to those found in the human and bovine PNP structures. The enzymatic activity assay of givPNP on various substrates revealed that givPNP can only accept 6-oxopurine nucleosides as substrates, which is also suggested by its aminoacid composition and active-site architecture. All these results suggest that givPNP is a homologue of mammalian PNPs in terms of amino-acid sequence, molecular mass, substrate specificity and overall structure, as well as in the composition of the active site.

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Characterization of the adenine nucleoside specific phosphorylase of Bacillus cereus

Cited by 7 publications

References 35 publications

Insights into Phosphate Cooperativity and Influence of Substrate Modifications on Binding and Catalysis of Hexameric Purine Nucleoside Phosphorylases

Insights into Phosphate Cooperativity and Influence of Substrate Modifications on Binding and Catalysis of Hexameric Purine Nucleoside Phosphorylases

Identification and Characterization of Two Adenosine Phosphorylase Activities in Mycobacterium smegmatis

Structure of grouper iridovirus purine nucleoside phosphorylase

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