Evolved Stereoselective Hydrolases for Broad-Spectrum G-Type Nerve Agent Detoxification

SummaryA preferred strategy for preventing nerve agents intoxication is catalytic scavenging by enzymes that hydrolyze them before they reach their targets. Using directed evolution, we simultaneously enhanced the activity of a previously described serum paraoxonase 1 (PON1) variant for hydrolysis of the toxic SP isomers of the most threatening G-type nerve agents. The evolved variants show ≤340-fold increased rates and catalytic efficiencies of 0.2-5 × 107 M−1 min−1. Our selection for prevention of acetylcholinesterase inhibition also resulted in the complete reversion of PON1's stereospecificity, from an enantiomeric ratio (E) < 6.3 × 10−4 in favor of the RP isomer of a cyclosarin analog in wild-type PON1, to E > 2,500 for the SP isomer in an evolved variant. Given their ability to hydrolyze G-agents, these evolved variants may serve as broad-range G-agent prophylactics

Molecular Toxicology Efficacy of the rePON1 mutant IIG1 to prevent cyclosarin toxicity

in vivo and to detoxify structurally different nerve agents in vitr

Design and in vitro realization of carbon-conserving photorespiration

Photorespiration recycles ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenation product, 2-phosphoglycolate, back into the Calvin Cycle. Natural photorespiration, however, limits agricultural productivity by dissipating energy and releasing CO$_2$. Several photorespiration bypasses have been previously suggested but were limited to existing enzymes and pathways that release CO$_2$. Here, we harness the power of enzyme and metabolic engineering to establish synthetic routes that bypass photorespiration without CO$_2$ release. By defining specific reaction rules, we systematically identified promising routes that assimilate 2-phosphoglycolate into the Calvin Cycle without carbon loss. We further developed a kinetic–stoichiometric model that indicates that the identified synthetic shunts could potentially enhance carbon fixation rate across the physiological range of irradiation and CO$_2$, even if most of their enzymes operate at a tenth of Rubisco’s maximal carboxylation activity. Glycolate reduction to glycolaldehyde is essential for several of the synthetic shunts but is not known to occur naturally. We, therefore, used computational design and directed evolution to establish this activity in two sequential reactions. An acetyl-CoA synthetase was engineered for higher stability and glycolyl-CoA synthesis. A propionyl-CoA reductase was engineered for higher selectivity for glycolyl-CoA and for use of NADPH over NAD$^+$, thereby favoring reduction over oxidation. The engineered glycolate reduction module was then combined with downstream condensation and assimilation of glycolaldehyde to ribulose 1,5-bisphosphate, thus providing proof of principle for a carbon-conserving photorespiration pathway