Process Biochemistry 47 (2012) 2219–2226 Contents lists available at SciVerse ScienceDirect Process Biochemistry jo u rn al hom epage: www.elsevier.com/locate/procbio Redesigning the active site of a carboxyl esterase from the archaeon Archaeoglobus fulgidus to improve sensitivity to organophosphorus compounds Yuexi Wang a,b , Binchun Li a,b , Weiwei Han b , Guangyu Yang a , Zuoming Zhang b , Yan Feng a,b,∗ a State Key Laboratory of Microbial Metabolism, College of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China b Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130021, China a r t i c l e i n f o Article history: Received 26 February 2012 Received in revised form 24 August 2012 Accepted 25 August 2012 Available online 5 September 2012 Keywords: Acetylcholinesterase Archaeoglobus fulgidus Thermophilic esterase Organophosphorus compound Mutagenesis Inhibition a b s t r a c t Organophosphorus compounds (OPs) are widely used as pesticides because of their ability to inhibit the activity of acetylcholinesterase (AChE) in the nervous system. Thus, AChE is generally used as a biosen- sor for pesticide detection. Due to the instability of AChE a more stable enzyme would be desirable for robust applications. We investigated the sensitivity of a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus (AFEST) to seven selected OPs. The IC 50 of dichlorvos against AFEST (50.8 ± 2.6 nM) was 10-fold lower than that of the commercially obtained AChE, indicating that AFEST had higher sensi- tivity. Its sensitivity for the other OPs was lower than AChE. To enhance the sensitivity of AFEST to OPs, site-directed mutations were introduced in the cap domain of AFEST. The sensitivity of mutant N44S/S48V was enhanced toward all seven OPs compared to the wild-type and was higher than AChE for four OPs, including paraoxon (3.3 ± 0.01 nM), dichlorvos (28.0 ± 0.6 nM), profenofos (43.0 ± 1.0 nM), and diazinon (3.0 ± 0.2 nM). The half-lives of AFEST and the mutant N44S/S48V at 37 ◦ C were over 15 d. The interactions between the enzymes and select OPs were investigated by molecular docking. The results demonstrated that AFEST and the mutant N44S/S48V have the potential to be biosensor for OP detection. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction Organophosphorus compounds (OPs) are one of the largest groups of pesticides and are widely used for crop protection in agriculture [1,2], because they are toxic to insects by virtue of their ability to inactivate acetylcholinesterase (AChE) [3–6]. How- ever, the overuse of OPs can also cause environmental pollution and lead to serious clinical and public health problems [7,8]. Thus, the exploration of enzymes for detecting OPs is an important focus for biologists [9,10]. Currently, mammalian AChE is the most fre- quently used biological agent for detecting OPs [3–6,11]. AChE hydrolyzes the substrate acetylcholine (ACh), and the catalytic activity is inhibited by OPs [12–14]. A cholinesterase biosensor has been developed according to this feature, which can be used to mea- sure the concentration of OPs. Recently, several serine hydrolases involved in the nervous system, including esterases and pepti- dases, were shown to be inhibited by OPs. In contrast to AChE, these enzymes exhibited different inhibitory spectra. Acylpeptide hydrolase (APH) from porcine brain was found to be more sensitive ∗ Corresponding author at: State Key Laboratory of Microbial Metabolism, College of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. Tel.: +86 21 34207189; fax: +86 21 34207189. E-mail address: yfeng2009@sjtu.edu.cn (Y. Feng). towards chlorpyrifos, dichlorvos, 3 H-diisopropyl fluorophosphate (DFP), and mipafox than AChE [15,16]. The neuropathy target esterase (NTE) exhibited higher sensitivity toward mipafox than AChE [17]. In addition, the fatty acid amide hydrolase (FAAH) from rat brain was found to be more sensitive towards octyl-P(O)Cl 2 , octyl-P(O)F 2 , and octyl-P(S)Cl 2 [18]. Human lysophosphatidyl- choline hydrolase is sensitive towards OPs with long alkyl groups, but is not very sensitive towards common OPs, such as paraoxon and dichlorvos [19]. Importantly, all of these described mammalian proteins exhibit different sensitivities to OPs and could potentially be used to detect the compounds [20]. However, the use of mam- malian enzymes as biosensors is limited for large scale applications due to low stability in vitro. It is expected that a recombinant enzyme and its mutants may possess the desired sensitivity and stability as demanded by a new biosensor. Thus, further studies are needed to identify more stable and sensitive detection enzymes. Thermophilic enzymes from eubacteria and archaea with extreme biological stability have the potential to be used as detectors. Recently, Manco et al. reported a thermophilic car- boxylesterase from Alicyclobacillus acidocaldarius (EST2) that can be irreversibly inhibited by paraoxon and methyl paraoxon [21–23]. A thermostable carboxylesterase from the hyperthermophilic archaeon Archaeoglobus fulgidus (AFEST) was also reported to belong to the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family, which has high homology to 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.08.021