Comparative investigation of the reaction mechanisms of the organophosphate-degrading phosphotriesterases from Agrobacterium radiobacter (OpdA) and Pseudomonas diminuta (OPH)

Abstract

Metal ion-dependent, organophosphate-degrading enzymes have acquired increasing attention due to their ability to degrade and thus detoxify commonly used pesticides and nerve agents such as sarin. The best characterized of these enzymes are from Pseudomonas diminuta (OPH) and Agrobacterium radiobacter (OpdA). Despite high sequence homology (>90 % identity) and conserved metal ion coordination these enzymes display considerable variations in substrate specificity, metal ion affinity/preference and reaction mechanism. In this study, we highlight the significance of the presence (OpdA) or absence (OPH) of an extended hydrogen bond network in the active site of these enzymes for the modulation of their catalytic properties. In particular, the second coordination sphere residue in position 254 (Arg in OpdA, His in OPH) is identified as a crucial factor in modulating the substrate preference and binding of these enzymes. Inhibition studies with fluoride also support a mechanism for OpdA whereby the identity of the hydrolysis-initiating nucleophile changes as the pH is altered. The same is not observed for OPH.

Enzimas degradantes de organofosforados: Base molecular e perspectivas para biorremediação enzimática de agroquímicos

“…The most important amino acids sequence differences between OpdA and OPH consist in: (1) different residues in the active site as Arg254/His254, Tyr257/His257 and Phe272/Leu272, respectively in OpdA and OPH (Pedroso et al, 2014), and (2) 20 additional amino acids at the C-terminus of OpdA, which seem to be irrelevant for catalysis since are located reasonably far away from the active site (Ely et al, 2010;Horne et al, 2006).…”

Kinetic, mechanistic, structural and spectroscopic investigations of Bimetallic Metallohydrolases

“…Fluoride is a potent inhibitor for a number of metal ion-dependent hydrolases, including the di-Ni 2+ urease (196) , the di-Mn 2+ arginase (197,198) and the Fe 3+ -M 2+ purple acid phosphatase (PAP, where M = Fe, Zn or Mn) (199,200) . OpdA is also inhibited by fluoride (Ki ~ 300 nM at pH 6.5), and the uncompetitive mode of inhibition, together with a strong exchange coupling of the metal ions is consistent with fluoride displacing the hydrolysis-initiating μ-OH (195) . Considering that OPH is expected to employ a mechanism similar to that of OpdA (vide supra) it is surprising that this enzyme is not inhibited by fluoride (195) .…”

“…OpdA is also inhibited by fluoride (Ki ~ 300 nM at pH 6.5), and the uncompetitive mode of inhibition, together with a strong exchange coupling of the metal ions is consistent with fluoride displacing the hydrolysis-initiating μ-OH (195) . Considering that OPH is expected to employ a mechanism similar to that of OpdA (vide supra) it is surprising that this enzyme is not inhibited by fluoride (195) . …”

“…Site-directed mutagenesis and in vitro evolution studies demonstrated that in particular two residues in the substrate binding pocket may play an important role in mediating these variations: while R254 and Y257 in OpdA are integrated into an extensive hydrogen bonding network that connects the substrate binding pocket to the metal ion center ( Figure. 3.2 A), the corresponding residues in OPH are two histidines, and no hydrogen bond network is present ( Figure 3.2 B). In OpdA this network controls the conformation of R254: when present the side chain is kinked, allowing access to the metal center, when absent the side chain is more linear, obstructing access (195) . It is this sequestration of the catalytic site that may underlie (i) the preference of OpdA for smaller substrates, (ii) this enzyme's enhanced catalytic efficiency and (iii) its increased metal ion affinity when compared to OPH (195) .…”

“…In OpdA this network controls the conformation of R254: when present the side chain is kinked, allowing access to the metal center, when absent the side chain is more linear, obstructing access (195) . It is this sequestration of the catalytic site that may underlie (i) the preference of OpdA for smaller substrates, (ii) this enzyme's enhanced catalytic efficiency and (iii) its increased metal ion affinity when compared to OPH (195) . This interpretation may also account for the distinct variation observed between OpdA and OPH with respect to their interaction with fluoride.…”