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OPHC2 is a thermostable organophosphate (OP) hydrolase in the β-lactamase superfamily. OPs are highly toxic synthetic chemicals with no natural analogs. How did OPHC2 acquire phosphotriesterase (PTE) activity remained unclear. In this study, an OPHC2 analogue, PoOPH was discovered from Pseudomonas oleovorans exhibiting high lactonase and esterase activities and latent PTE activity. Sequence analysis revealed conserved His250 and Ile263 and site-directed mutagenesis at these crucial residues enhanced PTE activity. The best variant PoOPHM2 carrying H250I/I263W mutations displayed 6,962- and 106-fold improvements in catalytic efficiency for methyl-parathion and ethyl-paraoxon degradation, whereas the original lactonase and esterase activities decreased dramatically. A 1.4 × 10(7) -fold of specificity inversion was achieved by only two residue substitutions. Significantly, thermostability of the variants was not compromised. Crystal structure of PoOPHM2 was determined at 2.25 Å resolution and docking studies suggested that the two residues in the binding pocket determine substrate recognition. Lastly, new organophosphorus hydrolases (OPHs) were discovered using simple double mutations. Among them, PpOPHM2 from Pseudomonas putida emerged as a new promising OPH with very high activity (41.0 U mg(-1) ) toward methyl-parathion. Our results offer a first scrutiny to PTE activity evolution of OPHs in β-lactamase superfamily and provide efficient and robust enzymes for OP detoxification.

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Bismuth is widely used in metallurgy, cosmetic industry, and medical diagnosis and recently, bismuth nanoparticles (NPs) (BiNP) have been made and proved to be excellent CT imaging agents. Previously, we have synthesized bovine serum albumin based BiNP for imaging purpose but we found a temporary kidney injury by BiNP. Due to the reported adverse events of bismuth on human health, we extended our studies on the mechanisms for BiNP induced nephrotoxicity. Blood biochemical analysis indicated the increase in creatinine (CREA) and blood urea nitrogen (BUN), and intraluminal cast formation with cell apoptosis/necrosis was evident in proximal convoluted tubules (PCTs) of mice. BiNP induced acute kidney injury (AKI) was associated with an increase in LC3II, while the autophagic flux indicator p62 remained unchanged. Chloroquine and rapamycin were used to evaluate the role of autophagy in AKI caused by BiNP. Results showed that BiNP induced AKI was further attenuated by rapamycin, while AKI became severe when chloroquine was applied. In vitro studies further proved BiNP induced autophagy in human embryonic kidney cells 293, presented as autophagic vacuole (AV) formation along with increased levels of autophagy-related proteins including LC3II, Beclin1, and Atg12. Specifically, reactive oxygen species (ROS) generated by BiNP could be the major inducer of autophagy, because ROS blockage attenuated autophagy. Autophagy induced by BiNP was primarily regulated by AMPK/mTOR signal pathway and partially regulated by Akt/mTOR. Our study provides fundamental theory to better understand bismuth induced nephrotoxicity for better clinical application of bismuth related compounds.

Malathion is one of the most widely used organophosphorus pesticides in the United States and developing countries. Herein, we enhanced the degradation rate of malathion starting with a phosphotriesterase PoOPHM2 while also considering thermostability. In the first step, iterative saturation mutagenesis at residues lining the binding pocket (CASTing) was employed to optimize the enzyme active site for substrate binding and activity. Hot spots for enhancing activity were then discovered through epPCR-based random mutagenesis, and these beneficial mutations were then recombined by DNA shuffling. Finally, guided by in silico energy calculations (FoldX), thermostability of the variant was improved. The mutations extend from the core region to the enzyme surface during the evolutionary pathway. After screening <9,000 mutants, the best variant PoOPHM9 showed 25-fold higher activity than wild-type PoOPHM2 , with a thermostability (T50 (15) ) of 67.6°C. Thus, PoOPHM9 appears to be an efficient and robust candidate for malathion detoxification. Biotechnol. Bioeng. 2016;113: 2350-2357. © 2016 Wiley Periodicals, Inc.

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