The OP (organophosphate)-degrading enzyme from Agrobacterium radiobacter (OpdA)
is a binuclear metallohydrolase able to degrade highly toxic OP pesticides and
nerve agents into less or non-toxic compounds. In the present study, the effect
of metal ion substitutions and site-directed mutations on the catalytic
properties of OpdA are investigated. The study shows the importance of both the
metal ion composition and a hydrogen-bond network that connects the metal ion
centre with the substrate-binding pocket using residues Arg254 and Tyr257 in the
mechanism and substrate specificity of this enzyme. For the Co(II) derivative of
OpdA two protonation equilibria (pKa1 ~5; pKa2 ~10) have been identified as
relevant for catalysis, and a terminal hydroxide acts as the likely
hydrolysis-initiating nucleophile. In contrast, the Zn(II) and Cd(II)
derivatives only have one relevant protonation equilibrium (pKa ~4-5), and the
μOH is the proposed nucleophile. The observed mechanistic flexibility may
reconcile contrasting reaction models that have been published previously and
may be beneficial for the rapid adaptation of OP-degrading enzymes to changing
environmental pressures.
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