title: Efficient Fludarabine-Activating PNP From Archaea as a Guidance for Redesign the Active Site of E. Coli PNP.
abstract: The combination of the gene of purine nucleoside phosphorylase (PNP) from Escherichia coli and fludarabine represents one of the most promising systems in the gene therapy of solid tumors. The use of fludarabine in gene therapy is limited by the lack of an enzyme that is able to efficiently activate this prodrug which, consequently, has to be administered in high doses that cause serious side effects. In an attempt to identify enzymes with a better catalytic efficiency than E. coli PNP towards fludarabine to be used as a guidance on how to improve the activity of the bacterial enzyme, we have selected 5'-deoxy-5'-methylthioadenosine phosphorylase (SsMTAP) and 5'-deoxy-5'-methylthioadenosine phosphorylase II (SsMTAPII), two PNPs isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. Substrate specificity and catalytic efficiency of SsMTAP and SsMTAPII for fludarabine were analyzed by kinetic studies and compared with E. coli PNP. SsMTAP and SsMTAPII share with E. coli PNP a comparable low affinity for the arabinonucleoside but are better catalysts of fludarabine cleavage with k(cat)/K(m) values that are 12.8-fold and 6-fold higher, respectively, than those reported for the bacterial enzyme. A computational analysis of the interactions of fludarabine in the active sites of E. coli PNP, SsMTAP, and SsMTAPII allowed to identify the crucial residues involved in the binding with this substrate, and provided structural information to improve the catalytic efficiency of E. coli PNP by enzyme redesign.
authors.affiliation: Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Via L. De Crecchio 7, Naples, 80138, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Cacciapuoti
authors.affiliation: Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Via L. De Crecchio 7, Naples, 80138, Italy.
authors.forename: Maria Libera
authors.initials: ML
authors.lastname: Bagarolo
authors.affiliation: Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Via L. De Crecchio 7, Naples, 80138, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Martino
authors.affiliation: Department of Chemistry and Biology, University of Salerno, Via Giovanni Paolo II 132, Fisciano, Salerno, 84084, Italy.
authors.forename: Bernardina
authors.initials: B
authors.lastname: Scafuri
authors.affiliation: Department of Chemistry and Biology, University of Salerno, Via Giovanni Paolo II 132, Fisciano, Salerno, 84084, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Marabotti
authors.affiliation: Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Via L. De Crecchio 7, Naples, 80138, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Porcelli
mesh_terms: D000241:Adenosine / Q000737:chemistry / Q000378:metabolism; D001087:Arabinonucleosides / Q000737:chemistry / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D000072417:Protein Domains; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology*; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: D001087:Arabinonucleosides; D019843:Archaeal Proteins; D029968:Escherichia coli Proteins; D007527:Isoenzymes; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase; D014740:Vidarabine; D000241:Adenosine; C024352:fludarabine
keywords: FLUDARABINE; GENE THERAPY; MOLECULAR DOCKING; PURINE NUCLEOSIDE PHOSPHORYLASE; SsMTAP; SsMTAPII