Introduction

1. Figure 2 DHAP (-hydroxyketone) can be seen as a molecule with a catalytic head-group (red box; R) and an anchor moiety (green box A). In wild type the anchor triggers the catalytic machinery, after which catalysis occurs. Tailored ligands are based on this principle, where R is unchanged. Figure 1 Figure 3 Conceptual overview of interactions: striped boxes represent historic linage of TIM variants, blue represents (random) mutagenesis experiments, white boxes are additional and available resources, and purple are deliverables. Wild type TIM concerns trypanosomal TIM. Schematic representation of the phage display technique in which immobilized suicide inhibitors will capture reactive A-TIM variants, by covalently binding to the catalytic glutamate. The inset in the right bottom corner shows a schematic drawing of the mode of binding of citric acid in a 1.5 Å structure of A-TIM. Non-natural enzymes: Directed evolution of a monomeric triosephosphate isomerase variant – a case study Mari Ylianttila1#, Marco G. Casteleijn1, Markus Alahuhta2, Matti Vaismaa3, Ritva Juvani3, Sampo Mattila3, Marja Lajunen3, Jouni Pursiainen3, Kristiina Takkinen4, Rik K. Wierenga2, Peter Neubauer1. 1) University of Oulu (Finland), Bioprocess Engineering Laboratory, Dept. of Process and Environm. Engineering; 2) University of Oulu (Finland), Dept. of Biochemistry; 3) University of Oulu (Finland), Dept. of Chemistry, 4) University of Oulu, VTT Biotechnology, (Finland), # corresponding author: mari.ylianttila@oulu.fi Introduction Triosephosphate isomerase (TIM [1, 2]) is a glycolytic enzyme with high substrate specificity catalyzing only the interconversion of dihydroxyacetone phosphate (DHAP; -hydroxyketone) and D-glyceraldehyde-3-phosphate (DGAP; α-hydroxyaldehyde). The wild type enzyme is a dimer, and each subunit has the classical TIM-barrel fold. Our enzyme platform is based on the scaffold of a monomeric form of TIM which was engineered to have a deeper and wider binding groove (A-TIM). In figure 1 DHAP can be seen, where the molecule is methodologically divided in the changeable anchor moiety and the reactive end. A-TIM in directed evolution Tailored enzymes for tailored ligands • A-TIM is an ideal test case for directed evolution approaches aiming at fine tuning its catalytic properties, because it is • Small in size (suitable for NMR) • Monomeric protein • Easily crystallizable • Soluble and highly expressed in E. coli • No cofactors needed • Its wild type precursor is extensively studied TIM • Its broadened and deep widened binding pocket is able to bind new kind of substrates when compared to wild type TIM • Additionally, also the ICM docking technology and binding studies (with NMR, mass spectrometry, X-ray and surface plasma resonance) have identified several promising ‘lead’-compounds from a pool of 35 novel compounds which are candidate molecules for this project • Wild Type TIM is well understood, and many publications can be found on its catalytic and structural properties • The binding site is an extended groove • Knowledge of TIM properties will be continuously implemented in the protein engineering efforts. Directed enzyme evolution has been already tested for monoTIM with successful results [3]. The objective is to implement directed evolution and screening approaches, aiming at tailoring the properties of monomeric TIM such that it binds new substrates and becomes an efficient catalyst in the conversion of -hydroxyketones and chiral α-hydroxyaldehydes. The main technique to obtain the goals set in figure 2 can be seen in figure 3. Suicide inhibitors based on known “binders” will be used in the first step. Step 1: Substrate binding loops of TIM are modified by restricted random mutagenesis to obtain mutant enzymes which bind new kind of substrates. Phage display techniques will be used for screening additional “binders”. Step 2: Catalytic properties of the captured TIM variants will be improved by directed evolution approaches to become active catalysts. Active enzymes catalyzing the conversion of -hydroxyketones into chiral - hydroxyaldehydes will be screened by NMR. Such active enzyme will be called Kealases Conclusions • Monomeric TIM is an excellent scaffold for enzyme engineering • Bound tailored ligands (“binders”) are ideal lead compounds to start site directed evolution • Monomeric TIM which convert -hydroxyketones will be called Kealases References Acknowledgements This work is supported by the Academy of Finland (project 53923), Tekes (Biocatnuc), European Union Structural Funds and State Provincial Office of Oulu. • Wierenga R.K. (2001) The TIM-barrel fold: a versatile framework for efficient enzymes. FEBS Lett 492, 193-198. • Kursula, I. and Wierenga, R.K. (2003) Crystal structure of triosephosphate isomerase complexed with 2-phospho-glycolate at 0.83Å resolution. J Biol Chem 278, 9544-955. • Saab-Rincon G., Juarez V.R., Osuna J., Sanchez F., Soberon X. (2001). Different strategies to recover the activity of monomeric triosephosphate isomerase by directed evolution. Protein Eng 14, 149-155. • Borchert T.V., Abagyan R., Kishan K.V., Zeelen J.P., Wierenga R.K. (1993). The crystal structure of an engineered monomeric triosephosphate isomerase, monoTIM: the correct modelling of an eight-residue loop. Structure 1, 205-213. • Borchert T.V., Abagyan R., Jaenicke R., Wierenga R.K. (1994). Design, creation, and characterization of a stable, monomeric triosephosphate isomerase. Proc Natl Acad Sci USA 91, 1515-1518. • Norledge B.V., Lambeir A.M., Abagyan R.A., Rottmann A., Fernandez A.M., Filimonov V.V., Peter M.G., Wierenga R.K. 2001. Modelling, mutagenesis and structural studies on the fully conserved phosphate binding loop (loop-8) of triosephosphate isomerase: towards a new substrate specificity. Proteins 42, 383-389. • Thanki N., Zeelen J.Ph., Mathieu M., Jaenicke R., Abagyan R.A.A., Wierenga R.K., Schliebs W. (1997) Protein engineering with monomeric triophosphate isomerase (monoTIM): the modelling and structure verification of a seven-residue loop. 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