TY - JOUR
T1 - Crystal structure of purine nucleoside phosphorylase from Thermus thermophilus
AU - Tahirov, Tahir H.
AU - Inagaki, Eiji
AU - Ohshima, Noriyasu
AU - Kitao, Tomoe
AU - Kuroishi, Chizu
AU - Ukita, Yoko
AU - Takio, Koji
AU - Kobayashi, Masanori
AU - Kuramitsu, Seiki
AU - Yokoyama, Shigeyuki
AU - Miyano, Masashi
N1 - Funding Information:
We are grateful to Maki Kumei for assistance with the crystallization, Mitsuaki Sugahara for management of the automated crystallization, Hitomi Takahashi for assistance with the preparation of the ligand containing solutions for crystal soaking, Naoko Takahashi and Mayumi Sugahara for protein characterization, Katsuhide Yutani for consultations in ITC measurements, and Masumi Maekawa for assistance with the functional assays. This work was supported by National Project on Protein Structural and Functional Analysis funded by MEXT of Japan (Project TT0127/HTPF00140).
PY - 2004/4/9
Y1 - 2004/4/9
N2 - The purine nucleoside phosphorylase from Thermus thermophilus crystallized in space group P43212 with the unit cell dimensions a=131.9Å, and c=169.9Å, and one biologically active hexamer in the asymmetric unit. The structure was solved by the molecular replacement method and refined at a 1.9Å resolution to an Rfree value of 20.8%. The crystals of the binary complex with sulfate ion and ternary complexes with sulfate and adenosine or guanosine were also prepared and their crystal structures were refined at 2.1Å, 2.4Å and 2.4Å, respectively. The overall structure of the T.thermophilus enzyme is similar to the structures of hexameric enzymes from Escherichia coli and Sulfolobus solfataricus, but significant differences are observed in the purine base recognition site. A base recognizing aspartic acid, which is conserved among the hexameric purine nucleoside phosphorylases, is Asn204 in the T.thermophilus enzyme, which is reminiscent of the base recognizing asparagine in trimeric purine nucleoside phosphorylases. Isothermal titration calorimetry measurements indicate that both adenosine and guanosine bind the enzyme with nearly similar affinity. However, the functional assays show that as in trimeric PNPs, only the guanosine is a true substrate of the T.thermophilus enzyme. In the case of adenosine recognition, the Asn204 forms hydrogen bonds with N6 and N7 of the base. While in the case of guanosine recognition, the Asn204 is slightly shifted together with the β9α7 loop and predisposed to hydrogen bond formation with O6 of the base in the transition state. The obtained experimental data suggest that the catalytic properties of the T.thermophilus enzyme are reminiscent of the trimeric rather than hexameric purine nucleoside phosphorylases.
AB - The purine nucleoside phosphorylase from Thermus thermophilus crystallized in space group P43212 with the unit cell dimensions a=131.9Å, and c=169.9Å, and one biologically active hexamer in the asymmetric unit. The structure was solved by the molecular replacement method and refined at a 1.9Å resolution to an Rfree value of 20.8%. The crystals of the binary complex with sulfate ion and ternary complexes with sulfate and adenosine or guanosine were also prepared and their crystal structures were refined at 2.1Å, 2.4Å and 2.4Å, respectively. The overall structure of the T.thermophilus enzyme is similar to the structures of hexameric enzymes from Escherichia coli and Sulfolobus solfataricus, but significant differences are observed in the purine base recognition site. A base recognizing aspartic acid, which is conserved among the hexameric purine nucleoside phosphorylases, is Asn204 in the T.thermophilus enzyme, which is reminiscent of the base recognizing asparagine in trimeric purine nucleoside phosphorylases. Isothermal titration calorimetry measurements indicate that both adenosine and guanosine bind the enzyme with nearly similar affinity. However, the functional assays show that as in trimeric PNPs, only the guanosine is a true substrate of the T.thermophilus enzyme. In the case of adenosine recognition, the Asn204 forms hydrogen bonds with N6 and N7 of the base. While in the case of guanosine recognition, the Asn204 is slightly shifted together with the β9α7 loop and predisposed to hydrogen bond formation with O6 of the base in the transition state. The obtained experimental data suggest that the catalytic properties of the T.thermophilus enzyme are reminiscent of the trimeric rather than hexameric purine nucleoside phosphorylases.
KW - Adenosine
KW - Guanosine
KW - NP-I, nucleoside phosphorylase-I
KW - PNP, purine nucleoside phosphorylase
KW - Purine nucleoside phosphorylase
KW - Structural genomics
KW - Thermus thermophilus
KW - bPNP, calf spleen (bovine) PNP
KW - hPNP, human PNP
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U2 - 10.1016/j.jmb.2004.02.016
DO - 10.1016/j.jmb.2004.02.016
M3 - Article
C2 - 15046984
AN - SCOPUS:11144355305
SN - 0022-2836
VL - 337
SP - 1149
EP - 1160
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 5
ER -