Structural basis of the substrate specificity of Bacillus cereus adenosine phosphorylase

Paola Dessanti, Yang Zhang, Simone Allegrini, Maria Grazia Tozzi, Francesco Sgarrella, Steven E. Ealick

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Purine nucleoside phosphorylases catalyze the phosphorolytic cleavage of the glycosidic bond of purine (2́-deoxy)nucleosides, generating the corresponding free base and (2́-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.

Original languageEnglish
Pages (from-to)239-248
Number of pages10
JournalActa Crystallographica Section D: Biological Crystallography
Volume68
Issue number3
DOIs
Publication statusPublished - Mar 2012

Fingerprint

Inosine
Purinones
Bacillus cereus
Substrate Specificity
Adenosine
Nucleosides
Sulfates
Catalytic Domain
Purine-Nucleoside Phosphorylase
Asparagine
Mutant Proteins
Hydrogen
Escherichia coli
Mutation
Water
Enzymes
adenosine phosphorylase
purine

Keywords

  • purine nucleoside phosphorylases
  • reversal of substrate preference
  • substrate specificity
  • transition-state stabilization

ASJC Scopus subject areas

  • Structural Biology

Cite this

Structural basis of the substrate specificity of Bacillus cereus adenosine phosphorylase. / Dessanti, Paola; Zhang, Yang; Allegrini, Simone; Tozzi, Maria Grazia; Sgarrella, Francesco; Ealick, Steven E.

In: Acta Crystallographica Section D: Biological Crystallography, Vol. 68, No. 3, 03.2012, p. 239-248.

Research output: Contribution to journalArticle

Dessanti, Paola ; Zhang, Yang ; Allegrini, Simone ; Tozzi, Maria Grazia ; Sgarrella, Francesco ; Ealick, Steven E. / Structural basis of the substrate specificity of Bacillus cereus adenosine phosphorylase. In: Acta Crystallographica Section D: Biological Crystallography. 2012 ; Vol. 68, No. 3. pp. 239-248.
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