Structural basis of the substrate specificity of Bacillus cereus adenosine phosphorylase

Abstract

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

Figure 1

Structure of the B. cereus AdoP hexamer in ribbon representation. Protomers B, C, D, E and F were generated by crystallographic symmetry from protomer A. Protomers that form two active sites at the interface are shown in similar colors (AB, CD and EF). Adenosine and sulfate are shown in red in stick representation.

Figure 2

Structure of the B. cereus AdoP protomer. The protomer of AdoP is shown in ribbon representation and colored by secondary structure. Adenosine and sulfate are shown in red stick representation.

Figure 3

Schematic diagram of the B. cereus AdoP active site. Residues belonging to the adjacent subunit are shown in red. Hydrogen bonds are indicated by dashed lines and labeled with donor-to-acceptor distances.

Figure 4

Comparison of Asp204/Asn204 in the four ternary complexes and the AdoP–SO₄ complex. The five structures are superimposed on the basis of secondary-structure element alignment. Color coding is red, AdoP–Ado–SO₄; orange, D204N–Ado–SO₄; blue, D204N–Ino–SO₄; green, AdoP–Ino–SO₄; cyan, AdoP–SO₄.

Figure 5

Stereo diagrams of the base-binding site. (a) AdoP–Ado–SO₄ complex. (b) AdoP–Ino–SO₄ complex. (c) D204N–Ado–SO₄ complex. (d) D204N–Ino–SO₄ complex. C atoms are shown in gray for the protein, in light blue for adenosine and in green for inosine. N atoms are shown in blue, O atoms in red and S atoms in yellow. Water molecules are shown as red spheres and labeled HOH. Hydrogen bonds are indicated by dashed lines and labeled with distances in Å.

Figure 6

Expected transition state for the phosphorolysis of adenosine by B. cereus AdoP.

Figure 7

Active-site stereoview of the AdoP–Ado–SO₄ and the E. coli PNP–Ado–PO₄ (PDB entry 1pk7; Bennett et al., 2003 ▶) complexes superimposed. Secondary structures are shown in ribbon representation and are colored yellow for B. cereus AdoP and light blue for E. coli PNP. C atoms are shown in green for B. cereus AdoP and in gray for E. coli PNP. N atoms are shown in blue, O atoms in red, S atoms in yellow and P atoms in magenta. Key residues are shown in stick representation and labeled in red for B. cereus AdoP and in blue for E. coli PNP. Hydrogen bonds are indicated by dashed lines.