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Construction of Genetically Engineered Recombinant Human Paraoxonase 1 (rHu PON1) Variants as Bioscavengers
Author(s) -
Chambers Carolyn,
Watson Cetara,
Pechik Igor
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.727.1
PON1 is a catalytic bioscavenger that has been studied for its ability to hydrolyze OP (organophosphorus) nerve agents such as soman and sarin, and its most commonly used substrate, paraoxon. Before Hu PON1 can be used as a catalytic bioscavenger of OPs in humans (a) the catalytic activity of native PON1 against OPs will have to be enhanced at least 10‐fold; (b) the association of PON1 with high density lipoprotein (HDL) will have to be disrupted while retaining stability of the enzyme; and (c) the uncharacteristically closed active‐site of PON1, which reduces the access of larger substrates to the active‐site, must be modified/opened/relaxed. Based on these issues, Hu PON1 suitable for use as a catalytic bioscavenger will likely be produced by recombinant DNA technology. Each recombinant protein was assayed for arylesterase and paraoxonase activities using substrates phenyl acetate or paraoxon, respectively. The initial levels for arylesterase activity were 0.1490 and 0.1417 units/mL, wild type (WT) and helix‐1 (H1) variant, and for paraoxonase activity were 0.2412 and 0.3388 units/mL, WT and H1 variant, respectively. The activity levels for helix‐2 variant using the substrate paraoxon ranged from 0.0755 to 0.1211 units/mL. Molecular modeling using paraoxon binding by variants revealed no substantial impact of the amino acid substitutions or helix replacement on the ligand positioning in the active site cleft.