Uttamkumar Samanta

University of Delaware, Newark, DE, United States

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Publications (4)14.28 Total impact

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    ABSTRACT: The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of P(R) and P(S) stereoisomers at the P-chiral center. The tabun complex displayed only the P(R) stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix-assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long-term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents.
    Biochemical pharmacology 05/2009; 78(4):420-9. · 4.25 Impact Factor
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    ABSTRACT: Insecticide and nerve agent organophosphorus (OP) compounds are potent inhibitors of the serine hydrolase superfamily of enzymes. Nerve agents, such as sarin, soman, tabun, and VX exert their toxicity by inhibiting human acetycholinesterase at nerve synapses. Following the initial phosphonylation of the active site serine, the enzyme may reactivate spontaneously or through reaction with an appropriate nucleophilic oxime. Alternatively, the enzyme-nerve agent complex can undergo a secondary process, called "aging", which dealkylates the nerve agent adduct and results in a product that is highly resistant to reactivation by any known means. Here we report the structures of paraoxon, soman, and sarin complexes of group-VIII phospholipase A2 from bovine brain. In each case, the crystal structures indicate a nonaged adduct; a stereoselective preference for binding of the P(S)C(S) isomer of soman and the P(S) isomer of sarin was also noted. The stability of the nonaged complexes was corroborated by trypsin digest and electrospray ionization mass spectrometry, which indicates nonaged complexes are formed with diisopropylfluorophosphate, soman, and sarin. The P(S) stereoselectivity for reaction with sarin was confirmed by reaction of racemic sarin, followed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate each stereoisomer. The P(S) stereoisomers of soman and sarin are known to be the more toxic stereoisomers, as they react preferentially to inhibit human acetylcholinesterase. The results obtained for nonaged complexes of group-VIII phospholipase A2 are compared to those obtained for other serine hydrolases and discussed to partly explain determinants of OP aging. Furthermore, structural insights can now be exploited to engineer variant versions of this enzyme with enhanced nerve agent binding and hydrolysis functions.
    Biochemistry 04/2009; 48(15):3425-35. · 3.38 Impact Factor
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    Uttamkumar Samanta, Cheryl Wilder, Brian J Bahnson
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    ABSTRACT: The plasma form of the human enzyme platelet activating factor acetylhydrolase (PAF-AH) has been crystallized, and X-ray diffraction data were collected at a synchrotron source to a resolution of 1.47 A. The crystals belong to space group C2, with unit cell parameters of a = 116.18, b = 83.06, c = 96.71 A, and beta= 115.09 degrees and two molecules in the asymmetric unit. PAF-AH functions as a general anti-inflammatory scavenger by reducing the levels of the signaling molecule PAF. Additionally, the LDL bound enzyme has been linked to atherosclerosis due to its hydrolytic activities of pro-inflammatory agents, such as sn-2 oxidatively fragmented phospholipids.
    Protein and Peptide Letters 02/2009; 16(1):97-100. · 1.99 Impact Factor
  • Uttamkumar Samanta, Brian J Bahnson
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    ABSTRACT: Human plasma platelet-activating factor (PAF) acetylhydrolase functions by reducing PAF levels as a general anti-inflammatory scavenger and is linked to anaphylactic shock, asthma, and allergic reactions. The enzyme has also been implicated in hydrolytic activities of other pro-inflammatory agents, such as sn-2 oxidatively fragmented phospholipids. This plasma enzyme is tightly bound to low and high density lipoprotein particles and is also referred to as lipoprotein-associated phospholipase A2. The crystal structure of this enzyme has been solved from x-ray diffraction data collected to a resolution of 1.5 angstroms. It has a classic lipase alpha/beta-hydrolase fold, and it contains a catalytic triad of Ser273, His351, and Asp296. Two clusters of hydrophobic residues define the probable interface-binding region, and a prediction is given of how the enzyme is bound to lipoproteins. Additionally, an acidic patch of 10 carboxylate residues and a neighboring basic patch of three residues are suggested to play a role in high density lipoprotein/low density lipoprotein partitioning. A crystal structure is also presented of PAF acetylhydrolase reacted with the organophosphate compound paraoxon via its active site Ser273. The resulting diethyl phosphoryl complex was used to model the tetrahedral intermediate of the substrate PAF to the active site. The model of interface binding begins to explain the known specificity of lipoprotein-bound substrates and how the active site can be both close to the hydrophobic-hydrophilic interface and at the same time be accessible to the aqueous phase.
    Journal of Biological Chemistry 10/2008; 283(46):31617-24. · 4.65 Impact Factor