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Structural basis of the substrate specificity of Bacillus cereus adenosine phosphorylase

Dessanti, Paola and Zhang, Yang and Allegrini, Simone and Tozzi, Maria Grazia and Sgarrella, Francesco and Ealick, Steven E. (2012) Structural basis of the substrate specificity of Bacillus cereus adenosine phosphorylase. Acta Crystallographica Section D, Vol. 68 (3), p. 239-248. eISSN 1399-0047. Article.

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DOI: 10.1107/S090744491200073X

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Abstract

Purine nucleoside phosphorylases catalyze the phosphorolytic cleavage of the glycosidic bond of purine (20-deoxy)nucleosides, generating the corresponding free base and (20-deoxy)- ribose 1-phosphate. Two classes of PNPs have been identified: homotrimers specific for 6-oxopurines and homohexamers that accept both 6-oxopurines and 6-aminopurines. Bacillus cereus adenosine phosphorylase (AdoP) is a hexameric PNP; however, it is highly specific for 6-aminopurines. To investigate the structural basis for the unique substrate specificity of AdoP, the active-site mutant D204N was prepared and kinetically characterized and the structures of the wild-type protein and the D204N mutant complexed with adenosine and sulfate or with inosine and sulfate were determined at high resolution (1.2–1.4 Å). AdoP interacts directly with the preferred substrate through a hydrogen-bond donation from the catalytically important residue Asp204 to N7 of the purine base. Comparison with Escherichia coli PNP revealed a more optimal orientation of Asp204 towards N7 of adenosine and a more closed active site. When inosine is bound, two water molecules are interposed between Asp204 and the N7 and O6 atoms of the nucleoside, thus allowing the enzyme to find alternative but less efficient ways to stabilize the transition state. The mutation of Asp204 to asparagine led to a significant decrease in catalytic efficiency for adenosine without affecting the efficiency of inosine cleavage.

Item Type:Article
ID Code:7568
Status:Published
Refereed:Yes
Uncontrolled Keywords:Purine nucleoside phosphorylases, substrate specificity, reversal of substrate preference, transition-state stabilization
Subjects:Area 05 - Scienze biologiche > BIO/10 Biochimica
Divisions:001 Università di Sassari > 01-a Nuovi Dipartimenti dal 2012 > Chimica e Farmacia
Publisher:Blackwell / Wiley
eISSN:1399-0047
Copyright Holders:© 2012 International Union of Crystallography
Deposited On:14 May 2012 13:06

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