Structure of bovine adenosine deaminase complexed with 6-hydroxy-1,6-dihydropurine riboside

Abstract

The crystal structure of adenosine deaminase (ADA) from bovine intestine complexed with a transition-state analogue, 6-hydroxy-1,6-dihydropurine riboside (HDPR), was solved at 2.5 A resolution by the molecular-replacement method using a homology model based on the crystal structure of mouse ADA. The final refinement converged to a crystallographic R factor of 20.7%. The C(alpha) backbone of bovine ADA is mostly superimposable on that of mouse ADA, although mouse ADA itself did not lead to a solution by molecular replacement. HDPR tightly interacts with ADA by means of six hydrogen bonds and is entirely enclosed within the active site. The lid of the envelope consists of two components: one contains two leucine residues, Leu55 and Leu59, and the other contains the backbone atoms Asp182 and Glu183. The C(delta) atoms of the two leucine residues are 3.5 A from the respective N atoms of the backbone. A weak interaction, similar to CH-pi binding, might make it possible to open the lid. Taking account of the movement and observation of this structural feature, the aim is to design novel ADA inhibitors.

Transition-State Variation in Human, Bovine, and Plasmodium falciparum Adenosine Deaminases

“…All solids were dissolved when the reaction was heated to 90 °C. The reaction mixture was then stirred under these conditions for another 12 h and volatile residues were removed under reduced pressure, followed by oil pump vacuum for 24 h. The solid residues were subject to recrystallization in hot water to give pure [6-13 C], [6][7][8][9][10][11][12][13][14][15] C]-labeled adenosines were prepared from radiolabeled riboses 7 and isotopically labeled adenines through two one-pot enzymatic reactions. Specifically labeled ATPs were synthesized as described previously with some modification.…”

“…Generation of Meisenheimer complex is followed by [ Table 2). The primary [6][7][8][9][10][11][12][13][14][15] (Figure 3). The hydroxyl attack and sp 3 rehybridization at C6 make the N6 electrostatic potential more negative in the transition states with overall charges of -0.912 to -0.925.…”

“…15,19,[24][25][26][27][28] Our interest in enzymatic transition states led us to determine the transition state structures of PfADA, HsADA and BtADA via the KIE approach. We also report a new synthetic method for making [6][7][8][9][10][11][12][13] C], [6][7][8][9][10][11][12][13][14][15] N], [6-13 C, 6-15 N]-labeled adenines. These adenines, together with [1-15 N] adenine, were combined with [1'-14 C] or [1'-3 H] riboses to give isotopically-labeled adenosines as substrates.…”

“…The set of KIEs for the three ADAs were determined under competitive conditions. The intrinsic [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] N], [6-13 C], and [6- 30 Adenylate kinase (AK), pyruvate kinase (PK), hexokinase, and bovine spleen adenosine deaminase (BtADA) were purchased from Sigma. Phospho-D-ribosyl-1-pyrophosphate (PRPP) synthase and adenine phosphoribosyltransferase (APRTase) were purified according to the methods reported previously.…”

Sampling protein motion and solvent effect during ligand binding

“…The binding of a ground-state inhibitor, like the hydroxylated form of adenosine (HDPR) (V in Fig. S1) (14), induces a conformational change that leads to the closed form. In this case, the gate is closed, the F1 and F2 sites are no longer accessible and the active site is composed only by S0 and F0 (Fig.…”