Enzyme Evolution: Exploring Substrate Scope and Catalytic Promiscuity I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Michael Fibinger, geb. Daumann geboren am 19.05.1988 in Berlin (Mitte) Greifswald, den 31.03.2016 Dekan: Prof. Dr. Werner Weitschies 1. Gutachter : Prof. Dr. Uwe T. Bornscheuer 2. Gutachter: Prof. Dr. Romas J. Kazlauskas Tag der Promotion: 27.06.2016 dedicated to my loving wife Diana I never predict anything, and I never will. Paul Gascoigne 3 Chapter 1. Exploring the enantioselective conversion of malonate esters by in silico modeling of the PLE isoenzymes Pig liver esterase (PLE) was first described in 1904 by its esterase activity in crude extracts of porcine liver.[1-2] The enzyme’s esterase activity is influenced by solvent addition and can dramatically increase, if crucial co-solvent concentrations are achieved.[3-8] First hypothesized as different subunits with separate enzymatic activity, today six isoenzymes (PLE1-6)[9-10] and a related alternative esterase[11] are known, differing only in a few amino acid residues. Each enzyme variant is able to form stable and active trimeric complexes. Small, but also bulky carboxylic esters, as well as thioesters or amides are accepted and hydrolyzed.[12] The broad substrate spectrum is caused by the intestinal function of detoxification and processing in pig liver, facing the complex composition of the omnivore diet. Sharing the same substrate specificity, the enantiomeric outcome can be very different throughout the isoenzymes. The achieved enantiomeric excess is solvent dependent, whereas the effect decreases with increasing size of solvent molecules.[7] Focusing this phenomenon, structural insights were obtained by molecular modeling and thus new residue contacts were found, explaining the binding mode of the investigated prochiral malonate diethyl esters. Part of this chapter has been published: M.E. Smith, M.P.C. Fibinger, U.T. Bornscheuer, D.S. Masterson, ChemCatChem 2015, 7, 3179-3185. doi: 10.1002/cctc.201500597 Chapter one 4 Content Introduction ......................................................................................................................... 5 PLE - a mixture of isoenzymes ......................................................................................... 5 Structural aspects of PLE ................................................................................................. 8 Properties of PLE and the stereoselective divergence ................................................. 12 Promiscuous PLE ........................................................................................................... 17 Results and Discussion ....................................................................................................... 18 Initial homology modeling of the PLE isoenzymes ....................................................... 18 Homology models of PLE2-6 and tunnel cluster analysis ............................................. 21 Docking preparation and former results ....................................................................... 27 Molecular docking of malonate esters ......................................................................... 28 Substrate 1 (diethyl 2-((1,3-dioxoisoindolin-2-yl)methyl)-2-methylmalonate) ....... 29 Substrate 2 (diethyl 2-((1,3-dithiooxoisoindolin-2-yl)methyl)-2-methylmalonate) . 33 Substrate 3 (diethyl 2-(benzyloxymethyl)-2-methylmalonate) ................................ 34 Substrate 4 (diethyl 2-(benzylthiomethyl)-2-methylmalonate) ............................... 36 Substrate 5 (diethyl 2-benzyl-2-methylmalonate) ................................................... 37 Substrate 6 (diethyl-2-methyl-2-((pyridine-2-yl-)methyl)malonate) ....................... 39 Substrate 7 (diethyl-2-methyl-2-((pyridine-3-yl-)methyl)malonate) ....................... 41 Substrate 8 (diethyl-2-methyl-2-((pyridine-4-yl-)methyl)malonate) ....................... 43 Molecular dynamics simulation of substrate 5 ............................................................. 46 Flexible residues and long-range interaction networks ................................................ 47 Concurring acidic residues of the catalytic triad ...................................................... 51 Summary ............................................................................................................................ 53 Materials and Methods ...................................................................................................... 54 Substrate preparation ................................................................................................... 54 Homology modeling ...................................................................................................... 54 Molecular docking ......................................................................................................... 54 Molecular dynamics simulation .................................................................................... 55 Tunnel cluster analysis .................................................................................................. 55 Network identification and residue contact detection ................................................. 55 Abbreviations ..................................................................................................................... 56 Reference ........................................................................................................................... 57 Chapter one 5 Introduction Pig liver esterase (PLE, also referred to as porcine liver esterase) is a broadly investigated enzyme of mammalian origin, accepting numerous carboxylic esters, even if these are sterically hindered or adjacently connected to bulky substituents. Then, and now, the broad substrate spectrum and the obtained regio- and enantioselectivity of PLE arouse high interest for the application of the enzyme in asymmetric synthesis or kinetic resolution of various esters to achieve enantiopure compounds (Figure 1). PLE, 25 °C pH 8, 1.5 h MeO2C CO2Me HOOC CO2Me NH2 94% NH2 ClCO2CH2Ph/Et3N 96% 1) PLE, 25 °C pH 8, 7 h MeO2C CO2Me 2) H2-Pd/C HOOC CO2Me NHCO2CH2Ph 93% NH2 Figure 1: Asymmetrization of 3-amino-pentanedioic acid dimethyl ester.[13] Both enantiomers are accessible by protection of the amino function with a bulky substituent, turning the enzyme’s selectivity towards the (S)-enantiomer. Subsequent ring closure yields a 4-membered lactam, the core function of the β-lactam antibiotic family of carbapenems. The first reported application of PLE was published in the beginning of the 20th century for the hydrolysis of mandelic esters[1-2], precursors for the drugs cyclandelate and homatropine. Later, PLE was successfully applied for the asymmetrization of prochiral mevalonic acids.[14] The rediscovery of the enzyme led to an enormous research interest, precisely because of its interesting features for organic synthesis and the increasing focus on enzymatic and chemoenzymatic catalysis. Despite the rising number of possible reactions involving PLE and the discovery of new substrate classes, its characterization was challenging. PLE - a mixture of isoenzymes In 1979, sixteen bands with esterase activity were recognized by electro-focusing and six major fractions with a pI from 4.8 to 5.8 were identified.[3] Analyzing the substrate specificity of these fractions, three major components were concluded.[4] They differ in their apparent molecular masses of 58.2 kDa (α-subunit), 59.7 kDa (β-subunit) and 61.4 kDa (γ-subunit) and their corresponding C-terminal amino acids leucine, glycine or alanine, respectively. Additionally, a lower aspartate, but higher arginine content was Chapter one: Introduction 6 found for the α-subunit compared to the γ-subunit. From the initial fractions, number I consisted mainly of the γ-subunit and exhibited choline esterase like properties, while accepting estrone acetate, butyrylcholine and long chain methanol esters. Fraction I is inhibited by fluoride ions and the parasympathomimetic (cholinomimetic) drug physostigmine. The pH optimum towards the conversion of methyl hexanoate was at 8.0. The organic co-solvents acetonitrile and acetone increased the enzymatic activity towards 4-nitrophenyl acetate. In case of the first co-solvent the rate doubles at a concentration of 1.25 mol L-1 acetonitrile. A maximum activity increase of 150% at 0.2 mol L-1 acetone was achieved, before the co-solvent inhibits the conversion at a concentration of 0.5 mol L-1 acetone. In contrast, methanol increases the phenol release from phenyl acetate 5-fold at a concentration of 1.25 mol L-1 methanol, but titrimetric measurements proved that the generated acetic acid undergoes transesterification by methyl ester formation. Fraction V contained the α-subunit and was not able to convert butyrylcholine, but short chain aliphatic esters. The highest rate of methyl hexanoate conversion was reached at pH 6.5. Solvents affected the activity of fraction V negatively: After a slight increase of conversion, concentrations higher than 2 mol L-1 acetonitrile inhibited the enzyme as well as minimal additions of acetone (25% residual activity at 0.2 mol L-1 acetone). Methanol increased the activity of fraction V only 1.7-fold. The reaction rates were measured with the same substrates