Directed evolution of hydrolases for prevention of G-type nerve agent intoxication

Abstract

Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of S(p)-cyclosarin by ∼10(5)-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with k(cat)/K(M) ∼ 10(7) M(-1) min(-1). We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

“…ultrahigh-throughput screening | microfluidic encapsulation | butyrylcholinesterase | Staphylococcus aureus | cell-cell interactions T he ultrahigh-throughput (1, 2) technique of screening (uHTS) in a double emulsion was applied in directed enzyme evolution (3,4) to investigate the idea that a universal genotypephenotype linkage was provided by compartmentalization. Artificial compartments of double emulsions were produced with high polydispersity by shear stress (5,6), which significantly decreased the portion of uniform droplets and thereby reduced the sensitivity and the maximal sorting rate.…”

Recent advances in enzyme promiscuity

“…However, there are evidences of different sub-sites within the same active site of enzymes. For example serum paraoxonase has native lactonase with promiscuous phosphotriesterase activity where hydrolysis of lactone is mediated by histidine dyad while phosphotriesterse activity is mediated by another set of residues in the same active site groove [1,3] (Fig. 2).…”

“…3) [3]. Based on the mutations appeared in the variants obtained by directed evolution experiment and PON1's active sites, we designed a library by incorporating synthetic oligonucleotides via gene reassembly.…”

“…The physiologically irrelevant promiscuous function of an enzyme is hypothesized to be the starting point for the evolution of new enzymes [1]. This hypothesis was proven by the excellently designed experiments in the laboratory by applying directed laboratory evolution technique on the targets like serum paraoxonase, bacterial phosphotriesterase and several other enzymes [3][4][5][6]. Subsequently, many other promiscuous enzymes were studied for the evolution of new enzymes [7,8], and to appreciate the mechanistic and evolutionary prospective [1,2,[9][10][11].…”

Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo

“…Further, the destruction of huge chemical warfare agents stocks may result in accidental poisoning to civilians and military personnel. In consequence, there is a clear demand for efficient, therapeutic anti-OP agent to overcome the adverse effects of drugs used for prophylaxis, emergency, and postexposure treatments (1)(2)(3).…”