Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.1 (phosphodiesterase)
 18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA-phosphate adducts are known to be formed by a variety of alkylating agents. Due to little or no repair of DNA-phosphate adducts, these adducts may offer increased possibilities of both identifying and quantifying DNA adducts. The formation of DNA-phosphate adducts leads to a complete esterification of the phosphate group giving rise to a phosphotriester configuration. This work consists of the characterization of ethyl phosphotriesters (Ethyl PTE) using miniaturized LC-ESI-MS/MS and column switching in enzymatic hydrolysate of DNA treated in vitro with the model compound N-ethyl-N-nitrosourea (ENU). In vitro ENU-treated DNA was enzymatically degraded using nuclease P1, phosphodiesterase, and alkaline phosphatase. The use of column switch allowed for large-volume injections, where unmodified nucleosides were discarded in the loading step. The analytes were forward flushed to the analytical column in the eluting step and separated using a linear gradient. Ten different ethyl PTEs (dGpEtdG, dApEtdA, dCpEtdC, TpEtT, dGpEtdA, dGpEtdC, dGpEtT, dApEtdC, dApEtT, and dCpEtT) were characterized by their masses and CAD product ion spectra. Measurements of accurate masses were carried out yielding experimental masses within 5 ppm of the calculated masses for 9 of the 10 ethyl PTEs. For comparison, the enzymatic hydrolysate of ENU-treated DNA was subjected to transalkylation of the DNA-phosphate adducts by cob(I)alamin. Formed ethyl-cobalamins were analyzed according to earlier developed methods. The limit of detection of an alkyl-cobalamin standard and an alkyl PTE standard was 2 fmol and 5 fmol, respectively.
 ...
PMID:Analysis of DNA-phosphate adducts in vitro using miniaturized LC-ESI-MS/MS and column switching: phosphotriesters and alkyl cobalamins. 1504 64

The mechanism by which the binuclear metallophosphotriesterases (PTEs, E.C. 3.1.8.1) catalyse substrate hydrolysis has been extensively studied. The mu-hydroxo bridge between the metal ions has been proposed to be the initiating nucleophile in the hydrolytic reaction. In contrast, analysis of some biomimetic systems has indicated that mu-hydroxo bridges are often not themselves nucleophiles, but act as general bases for freely exchangeable nucleophilic water molecules. Herein, we present crystallographic analyses of a bacterial PTE from Agrobacterium radiobacter, OpdA, capturing the enzyme-substrate complex during hydrolysis. This model of the Michaelis complex suggests the alignment of the substrate will favour attack from a solvent molecule terminally coordinated to the alpha-metal ion. The bridging of both metal ions by the product, without disruption of the mu-hydroxo bridge, is also consistent with nucleophilic attack occurring from the terminal position. When phosphodiesters are soaked into crystals of OpdA, they coordinate bidentately to the beta-metal ion, displacing the mu-hydroxo bridge. Thus, alternative product-binding modes exist for the PTEs, and it is the bridging mode that appears to result from phosphotriester hydrolysis. Kinetic analysis of the PTE and promiscuous phosphodiesterase activities confirms that the presence of a mu-hydroxo bridge during phosphotriester hydrolysis is correlated with a lower pK(a) for the nucleophile, consistent with a general base function during catalysis.
 ...
PMID:In crystallo capture of a Michaelis complex and product-binding modes of a bacterial phosphotriesterase. 1808 80

The recent specialization for utilization of pesticides reported for Pseudomonas diminuta phosphotriesterase (pPTE) strongly suggests that this activity evolved from an enzyme endowed with promiscuous phosphotriesterase activity. Such a putative "generalist" enzyme was recently proposed to be a member of the new phoshotriesterase-like lactonase family (PLL). The promiscuous carboxylesterase and phosphodiesterase activities detected in pPTE and PLLs in turn paved the way for the prediction of the existence in nature of PTE-like enzymes with predominant carboxylesterase or phosphodiesterase activities. An "in silico" analysis of the related Mesorhizobium loti ORF MLL7664 and the biochemical characterization demonstrated its prominent carboxylesterase and low phosphotriesterase specificity. On the basis of sequence similarity with the phosphotriesterase homology protein from Escherichia coli and the carboxylesterase activity, we called it phosphotriesterase-like carboxylesterase (MloPLC). The carboxylesterase activity is strictly dependent on divalent cations, and as such MloPLC is the first phosphotriesterase-like metal-carboxylesterase characterized to date. In related enzymes of the amidohydrolase superfamily either glutamate or carboxylated lysine substitutes for MloPLC glutamate 183 and the residue appear invariantly involved in maintaining the structural integrity of the binuclear metal center. Accordingly, we changed Glu-183 to lysine or glutamine. All the tested activities were completely abolished in the E183Q mutant, while only a residual phosphotriesterase activity could be detected in the E183K mutant. Surprisingly, in the latter mutant a parallel 650-fold specificity increase in bis-p-nitrophenyl-phosphate (BpNP-P) was observed, turning MloPLC from a carboxylesterase into a phosphodiesterase. Chemical, structural, and kinetic data strongly suggested that K183 is not carboxylated and that the gain of the new function is assisted by the substrate.
 ...
PMID:Evolution in the amidohydrolase superfamily: substrate-assisted gain of function in the E183K mutant of a phosphotriesterase-like metal-carboxylesterase. 1943 55