Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase^,

Abstract

Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k cat = 2.0 s −1 ; k cat /K m = 2.5 × 10 3 M −1 s −1 ). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn 2+ prior to induction, the purified enzyme was substantially more active for the deamination of adenine with values of k cat and k cat /K m of 200 s −1 and 5 × 10 5 M −1 s −1 , respectively. The apo-enzyme was prepared and reconstituted with Fe 2+ , Zn 2+ , or Mn 2+ . In each case, two enzyme-equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member within the deaminase sub-family of the amidohydrolase superfamily (AHS) to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe II /Fe II ]-ADE was oxidized to [Fe III /Fe III ]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe III /Fe III ]-ADE with dithionite restored the deaminase activity and thus the di-ferrous form of the enzyme is essential for catalytic activity. No evidence for spin-coupling between metal ions was evident by EPR or Mössbauer spectroscopies. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by Xray crystallography at 2.2 Å resolution and adenine was modeled into the active site based on homology to other members of the amidohydrolase superfamily. Based on the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH rate profiles and solvent viscosity were utilized to propose a chemical reaction mechanism and the identity of the rate limiting steps. † This work was supported in part by the National Institutes of Health (GM 71790, GM074945, and GM 46441). The X-ray coordinates and structure factors for Atu4426 have been deposited in the Protein Data Bank (PDB accession code: 3nqb) * To whom correspondence may be sent: (FMR) Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2012 March 22.Published in final edited form as: Biochemistry. 2011 March 22; 50(11): 1917-1927. doi:10.1021/bi101788n. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptAdenine deaminase (ADE 1 ) catalyzes the conversion of adenine to hypoxanthine and ammonia as shown in Scheme 1 (1,2). ADE is part of the purine degradation pathway where hypoxanthine is subsequently oxidized to uric acid by xanthine oxidase via a xanthine intermediate (3). This enzyme also participates in the purine salvage pathway for the synthesis of guanine nucleotides (1). ADE from Escherichia coli is a member of the amidohydrolase superfamily (AHS) and is clustered within cog1001 in ...

The catalase activity of diiron adenine deaminase

“…However, the diferrous form of ADE ec , made via reconstitution of apoprotein, is remarkably stable, even in the presence of O 2 . 2 In this article, we demonstrate that the extreme lability of ADE in the cell is due to the reaction of H 2 O 2 with the diferrous form of the enzyme, and suggest that this reaction may serve as a marker for oxidative stress in certain bacteria. [Fe II /Fe II ]-ADE catalyzes the disproportionation of H 2 O 2 to water and O 2 , and the formation of hydroxyl radical and superoxide from the same substrate.…”

“…2,3 However, the identity of the isolated protein could not be verified by mass spectrometry because multiple levels of post-translational modification made the spectrum impossible to interpret. Mass spectrometric analysis of the trypsin-generated fragments (70% coverage) identified the following residues as oxygenated: His-90, His-92, Met-97, Met-98, Met-186, Met-187, His-235, Met-251, Met-254, His-513, and Met-522 (data not shown).…”

“…1 The three-dimensional structure of ADE from Agrobacterium tumefaciens (ADE at ; locus tag: Atu4426) has been determined [Protein Data Bank (PDB) id: 3nqb] and the mechanism of action for the enzyme from Escherichia coli K12 (ADE ec ; locus tag: b3665) has been elucidated. 2 The ADEs from cog1001 are unique members of the AHS, since they require two divalent cations for maximal catalytic activity while the other deaminases from this superfamily require a single divalent cation in the active site. 1,2 The active site structure of ADE from A. tumefaciens is presented in Figure 1.…”

“…2 The ADEs from cog1001 are unique members of the AHS, since they require two divalent cations for maximal catalytic activity while the other deaminases from this superfamily require a single divalent cation in the active site. 1,2 The active site structure of ADE from A. tumefaciens is presented in Figure 1.…”

“…2 Unless precautions are taken to exclude the incorporation of iron into the enzyme during protein expression, the isolated enzyme becomes damaged and catalytic activity is severely compromised. However, the diferrous form of ADE ec , made via reconstitution of apoprotein, is remarkably stable, even in the presence of O 2 .…”