Publications

• Catalytic mechanism and three-dimensional structure of adenine deaminase.

  Kamat S.S., Bagaria A., Kumaran D., Holmes-Hampton G.P., Fan H., Sali A., Sauder J.M., Burley S.K., Lindahl P.A., Swaminathan S., Raushel F.M.

  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 sequest ... >> More

  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 k(cat) and k(cat)/K(m) values of 200 s(-1) and 5 × 10(5) M(-1) s(-1), respectively. The apoenzyme 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 of the deaminase subfamily of the amidohydrolase superfamily 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 diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Mössbauer spectroscopy. 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 on the basis of homology to other members of the amidohydrolase superfamily. On the basis of 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. << Less

  Biochemistry 50:1917-1927(2011) [PubMed] [EuropePMC]

• The catalase activity of diiron adenine deaminase.

  Kamat S.S., Holmes-Hampton G.P., Bagaria A., Kumaran D., Tichy S.E., Gheyi T., Zheng X., Bain K., Groshong C., Emtage S., Sauder J.M., Burley S.K., Swaminathan S., Lindahl P.A., Raushel F.M.

  Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided ... >> More

  Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn(2+) before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO(4). Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe(II) /Fe(II) ]-ADE catalyzed the conversion of H(2)O(2) to O(2) and H(2)O. The values of k(cat) and k(cat)/K(m) for the catalase activity are 200 s(-1) and 2.4 × 10(4) M(-1) s(-1), respectively. [Fe(II)/Fe(II)]-ADE underwent more than 100 turnovers with H(2)O(2) before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g(ave) = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H(2)O(2) by [Fe(II)/Fe(II)]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS. << Less

  Protein Sci 20:2080-2094(2011) [PubMed] [EuropePMC]

• The cryptic adenine deaminase gene of Escherichia coli. Silencing by the nucleoid-associated DNA-binding protein, H-NS, and activation by insertion elements.

  Petersen C., Moeller L.B., Valentin-Hansen P.

  In Escherichia coli there are two pathways for conversion of adenine into guanine nucleotides, both involving the intermediary formation of IMP. The major pathway involves conversion of adenine into hypoxanthine in three steps via adenosine and inosine, with subsequent phosphoribosylation of hypox ... >> More

  In Escherichia coli there are two pathways for conversion of adenine into guanine nucleotides, both involving the intermediary formation of IMP. The major pathway involves conversion of adenine into hypoxanthine in three steps via adenosine and inosine, with subsequent phosphoribosylation of hypoxanthine to IMP. The minor pathway involves formation of ATP, which is converted via the histidine pathway to the purine intermediate 5-amino-4-imidazolecarboxamide ribonucleotide and, subsequently, to IMP. Here we describe E. coli mutants, in which a third pathway for conversion of adenine to IMP has been activated. This pathway was shown to involve direct deamination of adenine to hypoxanthine by a manganese-dependent adenine deaminase encoded by a cryptic gene, yicP, which we propose be renamed ade. Insertion elements, located from -145 to +13 bp relative to the transcription start site, activated the ade gene as did unlinked mutations in the hns gene, encoding the histone-like protein H-NS. Gene fusion analysis indicated that ade transcription is repressed more than 10-fold by H-NS and that a region of 231 bp including the ade promoter is sufficient for this regulation. The activating insertion elements essentially eliminated the H-NS-mediated silencing, and stimulated ade gene expression 2-3-fold independently of the H-NS protein. << Less

  J. Biol. Chem. 277:31373-31380(2002) [PubMed] [EuropePMC]

• Adenine deaminase activity of the yicP gene product of Escherichia coli.

  Matsui H., Shimaoka M., Kawasaki H., Takenaka Y., Kurahashi O.

  During previous work on deriving inosine-producing mutants of Escherichia coli, we observed that an excess of adenine added to the culture medium was quickly converted to hypoxanthine. This phenomenon was still apparent after disruption of the known adenosine deaminase gene (add) on the E. coli ch ... >> More

  During previous work on deriving inosine-producing mutants of Escherichia coli, we observed that an excess of adenine added to the culture medium was quickly converted to hypoxanthine. This phenomenon was still apparent after disruption of the known adenosine deaminase gene (add) on the E. coli chromosome, suggesting that, like Bacillus subtilis, E. coli has an adenine deaminase. As the yicP gene of E. coli shares about 35% identity with the B. subtilis adenine deaminase gene (ade), we cloned yicP from the E. coli genome and developed a strain that overexpressed its product. The enzyme was purified from a cell extract of E. coli harboring a plasmid containing the cloned yicP gene, and had significant adenine deaminase [EC 3.5.4.2] activity. It was deduced to be a homodimer, each subunit having a molecular mass of 60 kDa. The enzyme required manganese ions as a cofactor, and adenine was its only substrate. Its optimum pH was 6.5-7.0 and its optimum temperature was 60 degrees C. The apparent Km for adenine was 0.8 mM. << Less

  Biosci. Biotechnol. Biochem. 65:1112-1118(2001) [PubMed] [EuropePMC]