title: Unique substrate specificity of purine nucleoside phosphorylases from Thermus thermophilus.
abstract: The degradation of purine nucleoside is the first step of purine nucleoside uptake. This degradation is catalyzed by purine nucleoside phosphorylase, which is categorized into two classes: hexameric purine nucleoside phosphorylase (6PNP) and trimeric purine nucleoside phosphorylase (3PNP). Generally, 6PNP and 3PNP degrade adenosine and guanosine, respectively. However, the substrate specificity of 6PNP and 3PNP of Thermus thermophilus (tt6PNP and tt3PNP, respectively) is the reverse of that anticipated based on comparison to other phosphorylases. Specifically, in this paper we reveal by gene disruption that tt6PNP and tt3PNP are discrete enzymes responsible for the degradation of guanosine and adenosine, respectively, in T. thermophilus HB8 cells. Sequence comparison combined with structural information suggested that Asn204 in tt6PNP and Ala196/Asp238 in tt3PNP are key residues for defining their substrate specificity. Replacement of Asn204 in tt6PNP with Asp changed the substrate specificity of tt6PNP to that of a general 6PNP. Similarly, substitution of Ala196 by Glu and Asp238 by Asn changed the substrate specificity of tt3PNP to that of a general 3PNP. Our results indicate that the residues at these positions determine substrate specificity of PNPs in general. Sequence analysis further suggested most 6PNP and 3PNP enzymes in thermophilic species belonging to the Deinococcus-Thermus phylum share the same critical residues as tt6PNP and tt3PNP, respectively.
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Tomoike
authors.forename: Seiki
authors.initials: S
authors.lastname: Kuramitsu
authors.forename: Ryoji
authors.initials: R
authors.lastname: Masui
mesh_terms: D000241:Adenosine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D006151:Guanosine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D006151:Guanosine; D011683:Purine-Nucleoside Phosphorylase; D000241:Adenosine
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