This journal is © The Royal Society of Chemistry 2017 Mol. BioSyst. Cite this: DOI: 10.1039/c7mb00196g Impact of phenylalanines outside the dimer interface on phosphotriesterase stability and function† Andrew J. Olsen,‡ a Leif A. Halvorsen,‡ b Ching-Yao Yang, a Roni Barak Ventura, a Liming Yin, a P. Douglas Renfrew, b Richard Bonneau bcd and Jin Kim Montclare * aefg We explore the significance of phenylalanine outside of the phosphotriesterase (PTE) dimer interface through mutagenesis studies and computational modeling. Previous studies have demonstrated that the residue-specific incorporation of para-fluorophenylalanine (pFF) into PTE improves stability, suggesting the importance of phenylalanines in stabilization of the dimer. However, this comes at a cost of decreased solubility due to pFF incorporation into other parts of the protein. Motivated by this, eight single solvent-exposed phenylalanine mutants are evaluated via ROSETTA and good correspondence between experiments and these predictions is observed. Three residues, F304, F327, and F335, appear to be important for PTE activity and stability, even though they do not reside in the dimer interface region or active site. While the remaining mutants do not significantly affect structure or activity, one variant, F306L, reveals improved activity at ambient and elevated temperatures. These studies provide further insight into role of these residues on PTE function and stability. Introduction Phosphotriesterase (PTE, E.C. 3.1.8.1) is an enzyme isolated from Pseudomonas diminuta capable of detoxifying organo- phosphorus agents (OPs). 1–7 OPs, which include pesticides 8,9 and chemical warfare agents, 10,11 cause hyper-stimulation inside synapses of the nervous system when covalently bound to the active site of acetylcholinesterase (AChE). 12 PTE hydrolyzes OPs via an S N 2-like reaction, preventing the adduct formation with AChE and subsequent inactivation. 13 PTE is composed of two (b/a) TIM-barrel subunits, each with a metallo-active site, and is only functional as a dimer. Various strategies have been employed to engineer PTE including rational mutagenesis, 14,15 computational design, 16,17 directed evolution, 18 and incorporation of non-canonical amino acids. 17,19 Using site-directed mutagenesis, the Raushel group investigated the impact of individual residues in the PTE active site 5,13,14,20,21 and was able to alter PTE specificity by generating single mutations G60A, I106A, F132A, and S308A in PTE. 14 Notably, the G60A mutant led to a hundredfold reduction in catalytic efficiency for dimethyl and diethyl p-nitrophenyl phos- phate, while I106A, F132A, and S308A demonstrated enhanced hydrolysis of R p -enantiomers. The steric constraints were relieved by substitution with the smaller size of amino acid. 14 These results demonstrated that PTE specificity could be modulated via mutations within the PTE active site. To identify hot spots for mutagenesis, Pavelka et al. developed the computational algorithm, HotSpot Wizard, integrated with structural, functional as well as evolutionary information, allowing quantification of mutability in the context of proteins. 16 After analysis, nine residues were identified for PTE mutagenesis: G60, L136, R139, S205, D235, A270, L271, L272, and F306. In comparison to experimental results from the Raushel group, G60 and F306 indeed played an important role in activity, affirming the hot spots of PTE. In the previous cases, residues targeted for mutagenesis have focused on the binding pocket or dimer interface. 14,22,23 However, there are examples from directed evolution experiments, a Department of Chemical and Biomolecular Engineering, New York University, Tandon School of Engineering, New York 11201, USA b Center for Genomics and Systems Biology, New York University, New York 10003, USA c Center for Computational Biology, Flatiron Institute, New York, NY 10010, USA d Courant Institute of Mathematical Sciences, Computer Science Department, New York University, New York, NY 10009, USA e Department of Chemistry, New York University, New York 10003, USA f Department of Biochemistry, SUNY Downstate Medical Center, Brooklyn 11203, USA g Department of Biomaterials, NYU College of Dentistry, New York 10010, USA. E-mail: montclare@nyu.edu; Fax: +1 (646) 997 3125; Tel: +1 (646) 997 3679 † Electronic supplementary information (ESI) available. See DOI: 10.1039/c7mb00196g ‡ These authors contributed equally. Received 1st April 2017, Accepted 7th August 2017 DOI: 10.1039/c7mb00196g rsc.li/molecular-biosystems Molecular BioSystems PAPER Published on 10 August 2017. Downloaded by New York University on 29/08/2017 02:52:26. View Article Online View Journal