Arch Toxicol DOI 10.1007/s00204-015-1626-2 MOLECULAR TOXICOLOGY Catalytic efficiencies of directly evolved phosphotriesterase variants with structurally different organophosphorus compounds in vitro Moshe Goldsmith2 · Simone Eckstein1 · Yacov Ashani2 · Per Greisen Jr.3 · Haim Leader4 · Joel L. Sussman5 · Nidhi Aggarwal5 · Sergey Ovchinnikov3 · Dan S. Tawfik2 · David Baker3 · Horst Thiermann1 · Franz Worek1 Received: 27 July 2015 / Accepted: 22 October 2015 © Springer-Verlag Berlin Heidelberg 2015 Abstract The nearly 200,000 fatalities following expo- catalytic efficiencies of V-agent hydrolysis by two newly sure to organophosphorus (OP) pesticides each year and selected PTE variants were determined. Moreover, in order the omnipresent danger of a terroristic attack with OP to establish trends in sequence–activity relationships along nerve agents emphasize the demand for the development of the pathway of PTE’s laboratory evolution, we examined effective OP antidotes. Standard treatments for intoxicated kcat/KM values of several variants with a number of V-type patients with a combination of atropine and an oxime are and G-type nerve agents as well as with different OP pes- limited in their efficacy. Thus, research focuses on develop- ticides. Although none of the new PTE variants exhibited 7 1 1 ing catalytic bioscavengers as an alternative approach using kcat/KM values >10 M− min− with V-type nerve agents, OP-hydrolyzing enzymes such as Brevundimonas diminuta which is required for effective prophylaxis, they were phosphotriesterase (PTE). Recently, a PTE mutant dubbed improved with VR relative to previously evolved variants. C23 was engineered, exhibiting reversed stereoselectivity The new variants detoxify a broad spectrum of OPs and and high catalytic efficiency (kcat/KM) for the hydrolysis of provide insight into OP hydrolysis and sequence–activity the toxic enantiomers of VX, CVX, and VR. Additionally, relationships. C23’s ability to prevent systemic toxicity of VX using a low protein dose has been shown in vivo. In this study, the Keywords Organophosphorus compounds · Organophosphate hydrolase · Phosphotriesterase · Bioscavenger · Mutant · Detoxification Moshe Goldsmith and Simone Eckstein have contributed equally to this article. Introduction Electronic supplementary material The online version of this article (doi:10.1007/s00204-015-1626-2) contains supplementary material, which is available to authorized users. Organophosphorus compounds (OPs) induce their toxic- ity by inhibition of the pivotal enzyme acetylcholinester- * Franz Worek ase (AChE). This results in accumulation of acetylcholine [email protected] (ACh) at central and peripheral synapses (Aldridge and 1 Bundeswehr Institute of Pharmacology and Toxicology, Reiner 1972; Marrs 2007) with subsequent overstimulation Neuherbergstrasse 11, 80937 Munich, Germany followed by multiple signs and symptoms, e.g., convul- 2 Department of Biological Chemistry, Weizmann Institute sions, respiratory arrest that leads eventually to death (Grob of Science, Rehovot, Israel 1956; Marrs 2007). The widespread use of OP pesticides 3 Institute for Protein Design and Department of Biochemistry, and the repeated misuse of OP nerve agents as chemical University of Washington, Seattle, WA 98195, USA warfare agents emphasize the need for an effective medical 4 Department of Materials and Interfaces, Weizmann Institute treatment. Currently, intoxication by OPs is treated with a of Science, Rehovot, Israel combination of atropine (muscarinic antagonist) and oxi- 5 Department of Structural Biology, Weizmann Institute mes (reactivation of inhibited AChE) (Johnson et al. 2000; of Science, Rehovot, Israel Thiermann et al. 2013); however, the standard therapy has 1 3 Arch Toxicol limited efficacy depending on the type and dose of OP and variant sequence determine its catalytic efficiency of (Worek and Thiermann 2013). hydrolysis. These disadvantages could be overcome by detoxifica- tion of OPs before entering target tissues (Nachon et al. 2013). An extensively investigated option is to use human Materials and methods butyrylcholinesterase (hBuChE), an enzyme which acts as a stoichiometric scavenger and therefore has to be applied Chemicals and reagents in high doses (Ashani and Pistinner 2004; Masson et al. 2009). An increasing focus is put on catalytic bioscav- OP nerve agents (Table 1) were made available by the engers, i.e., enzymes that hydrolyze OPs without being German Ministry of Defence. Stock solutions (1 or 0.1 % consumed and can be applied at low doses (Lenz et al. v/v) were prepared in acetonitrile and stored at ambi- 2007). Candidates are mammalian paraoxonase (PON1), ent temperature. OP pesticides (Table 1) were purchased Brevundimonas (formerly Pseudomonas diminuta), organo- from Dr. Ehrenstorfer GmbH (Augsburg, Germany). phosphate hydrolase (OPH) (also called phosphotriester- Stock solutions (10 % v/v) were prepared in acetonitrile ase—PTE), Alteromonas prolidase, organophosphorus acid and stored at ambient temperature. OP working solu- anhydrase (OPAA), and Loligo vulgaris diisopropylfluoro- tions were appropriately diluted in TNZ buffer (50 mM phosphatase (DFPase) (Masson and Rochu 2009). TRIS–HCl, pH 8.0, 100 mM NaCl, 10 µM ZnCl2) just OP nerve agents are racemic mixtures of the highly before use. 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) toxic P( ) and the less toxic P( ) enantiomer (de Jong and acetylthiocholine iodide (ATCh) were purchased from − + and Benschop 1988). Unfortunately, the P( ) enantiom- Sigma-Aldrich (Taufkirchen, Germany). All other chemi- − ers of nerve agents are barely hydrolyzed by the existing cals were purchased from Merck Eurolab (Darmstadt, wild-type enzymes due to their stereopreference toward Germany). Hemoglobin-free human erythrocyte ghosts the P( ) enantiomer (Bigley et al. 2013). Previous analysis served as AChE source and were prepared according to + estimated that effective in vivo detoxification using reason- (Dodge et al. 1963) with minor modifications (Bierwisch 1 able enzyme doses (<1 mg kg− body weight, assuming an et al. 2014). Restriction enzymes EcoRI-HF, PstI-HF and enzyme M.W. of ~40 kDa) demands a catalytic efficiency DpnI, pMAL-c2x expression vector and amylose beads 7 1 1 ® of >10 M− min− for hydrolysis of the toxic P( ) iso- were purchased from New England Biolabs (NEB ). − mers of nerve agents (Gupta et al. 2011). Hence, research Synthetic oligonucleotides were purchased from Sigma efforts are focused on engineering enzymes with high (Israel) Benzonase®nuclease from Novagen. Liquid and k /K values as well as P( ) stereopreference. solid bacterial growth media were purchased from the cat M − In addition, V-type nerve agents, e.g., VX, VR, and Bacteriology Unit (Weizmann Institute of Science, Reho- CVX, present a specific challenge due to their high percu- vot, Israel). taneous toxicity and long persistence in the human body (Masson et al. 2009). Recently, the engineered phosphotri- Computational library design esterase (PTE) mutant C23 with reversed stereoselectivity was found to hydrolyze the P( ) enantiomers of VX, CVX, A computational model of the active variant from ear- − 6 and VR with a catalytic efficiency, kcat/KM, up to 5 10 lier library screens, I_F4, was generated using the start- 1 1 × M− min− in vitro (Cherny et al. 2013) and was able to ing coordinates from the crystal structure of phosphotri- prevent systemic toxicity as post-exposure therapy in VX esterase from Pseudomonas diminuta with pdb accession poisoned guinea pigs at low protein dose (Worek et al. code 1HZY (Benning et al. 2001). The substitutions were 2014b). inserted into the initial structure and minimized using In the present study, we describe our attempts to obtain the all-atom energy function in Rosetta (Das and Baker recombinant PTE variants with improved catalytic efficien- 2008). cies for VX and VR hydrolysis using directed evolution To computationally screen substitutions at position methods. We provide a detailed in vitro analysis of the cat- C59, chain A of the crystal structure (PDB ID: 1HZY) alytic activities of two newly selected and four previously was used, and models with all 20 amino acids were gen- selected variants. The activities of these six PTE variants, erated using Rosetta. A computational 2D screen of the expressed and purified from E. coli cells, with 13 different positions A203X and D233X was performed using the V-type nerve agents, four G-type nerve agents, and three computational model of I_F4 as the starting model. Three pesticides are compared with that of the wild-type-like computational models were generated: one apo-structure PTE variant S5 (wt-PTE S5) (Roodveldt and Tawfik 2005). including the metal site as well as two holo structures with From these data, we attempt to explain how OP structure transition states models of VX and VR. All models were 1 3 Arch Toxicol Table 1 Chemical structures of tested OPs OP R1 R2 X Dimethyl-VX Methyl O-ethyl S-[2-(dimethylamino)ethyl] VM Methyl O-ethyl S-[2-(diethylamino)ethyl] n-Propyl-VX Methyl O-ethyl S-[2-(di-n-propylamino)ethyl] VX Methyl O-ethyl S-[2-(diisopropylamino)ethyl] CVX Methyl O-n-butyl S-[2-(diethylamino)ethyl] VR Methyl O-isobutyl S-[2-(diethylamino)ethyl] Methyl-VM Methyl O-methyl S-[2-(diethylamino)ethyl] Methyl-VE Ethyl O-ethyl S-[2-(dimethylamino)ethyl] VE Ethyl O-ethyl S-[2-(diethylamino)ethyl] Isopropyl-VE Ethyl O-ethyl S-[2-(diisopropylamino)ethyl] Amiton O-ethyl O-ethyl S-[2-(diethylamino)ethyl] Dimethyl-amiton O-ethyl O-ethyl S-[2-(dimethylamino)ethyl]