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 X-ray 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.