Mechanism and Structure of γ-Resorcylate Decarboxylase.

γ-Resorcylate decarboxylase (γ-RSD) has evolved to catalyze the reversible decarboxylation of 2,6-dihydroxybenzoate to resorcinol in a nonoxidative fashion. This enzyme is of significant interest because of its potential for the production of γ-resorcylate and other benzoic acid derivatives under environmentally sustainable conditions. Kinetic constants for the decarboxylation of 2,6-dihydroxybenzoate catalyzed by γ-RSD from Polaromonas sp. JS666 are reported, and the enzyme is shown to be active with 2,3-dihydroxybenzoate, 2,4,6-trihydroxybenzoate, and 2,6-dihydroxy-4-methylbenzoate. The three-dimensional structure of γ-RSD with the inhibitor 2-nitroresorcinol (2-NR) bound in the active site is reported. 2-NR is directly ligated to a Mn2+ bound in the active site, and the nitro substituent of the inhibitor is tilted significantly from the plane of the phenyl ring. The inhibitor exhibits a binding mode different from that of the substrate bound in the previously determined structure of γ-RSD from Rhizobium sp. MTP-10005. On the basis of the crystal structure of the enzyme from Polaromonas sp. JS666, complementary density functional calculations were performed to investigate the reaction mechanism. In the proposed reaction mechanism, γ-RSD binds 2,6-dihydroxybenzoate by direct coordination of the active site manganese ion to the carboxylate anion of the substrate and one of the adjacent phenolic oxygens. The enzyme subsequently catalyzes the transfer of a proton to C1 of γ-resorcylate prior to the actual decarboxylation step. The reaction mechanism proposed previously, based on the structure of γ-RSD from Rhizobium sp. MTP-10005, is shown to be associated with high energies and thus less likely to be correct.

Discovery of a cyclic phosphodiesterase that catalyzes the sequential hydrolysis of both ester bonds to phosphorus.

The bacterial C-P lyase pathway is responsible for the metabolism of unactivated organophosphonates under conditions of phosphate starvation. The cleavage of the C-P bond within ribose-1-methylphosphonate-5-phosphate to form methane and 5-phospho-ribose-1,2-cyclic phosphate (PRcP) is catalyzed by the radical SAM enzyme PhnJ. In Escherichia coli the cyclic phosphate product is hydrolyzed to ribose-1,5-bisphosphate by PhnP. In this study, we describe the discovery and characterization of an enzyme that can hydrolyze a cyclic phosphodiester directly to a vicinal diol and inorganic phosphate. With PRcP, this enzyme hydrolyzes the phosphate ester at carbon-1 of the ribose moiety to form ribose-2,5-bisphosphate, and then this intermediate is hydrolyzed to ribose-5-phosphate and inorganic phosphate. Ribose-1,5-bisphosphate is neither an intermediate nor a substrate for this enzyme. Orthologues of this enzyme are found in the human pathogens Clostridium difficile and Eggerthella lenta. We propose that this enzyme be called cyclic phosphate dihydrolase (cPDH) and be designated as PhnPP.

The catalase activity of diiron adenine deaminase.

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.

Catalytic mechanism and three-dimensional structure of adenine deaminase.

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.

Sensitivity and specificity improvement of an ion sensitive field effect transistors-based biosensor for potato glycoalkaloids detection.

Butyryl cholinesterase of different origin along with variations of the time of enzyme immobilization on the potentiometric transducer surface is offered to control the ion sensitive field effect transistor (ISFET)-based biosensor sensitivity. Because butyryl cholinesterase has been already used to develop the sensors for heavy metals, organophosphorus/carbamate pesticides, and steroidal glycoalkaloids analysis, the present study has been focused on the investigation and adjustment of the ISFET-based biosensor specificity exclusively to the glycoalkaloids. Utilization of ethylendiaminetetracetate (a complexon of heavy metal ions) and phosphotriesterase (a highly efficient catalyst for the hydrolysis of organophosphorus compounds) enabled the highly specific determination of glycoalkaloids at the background of lead and mercury (up to 500 microM of ions concentration) and paraoxon (up to 100 microM of pesticide concentration). The developed biosensor has been validated for glycoalkaloids detection in potato varieties cultivated in Ukraine, and the results obtained are compared to those measured by the methods of HPLC and TLC.