Towards new enzymes: understanding and engineering Marco G. Casteleijn 1#, Markus Alahuhta 2, Matti Vaismaa 3, Ritva Juvani 3, Ville Ratas 2, Sampo Mattila 3, Marja Lajunen 3, Jouni Pursiainen 3, Rik K. Wierenga 2, Peter Neubauer 1. 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. # corresponding author: Triose phosphate isomerase (TIM) is a glycolytic enzyme with very high substrate specificity. The wild type enzyme is a dimer, and each subunit has the classical TIM- barrel fold. Loops modifications resulted in a monomeric enzyme (monoTIM) [5] which remains active, although less so than the wild-type. Subsequently this has resulted in in a new variant (ml8bTIM) [5] with a much more extensive binding pocket which has no affinity for the original substrate. Our current studies attempt to change the substrate specificity of monomeric TIM. Our studies are focusing on understanding the catalytic properties of the Wild Type (Wt-TIM) [2, 3] and applying this knowledge to our newly engineered enzymes (fig 1). Introduction 1.Arkin M.R., Randal M, DeLano W.L., Hyde J., Luong T.N., Oslob J.D., Raphael D.R., Taylor L., Wang J., McDowell R.S., Wells J.A., Braisted A.C. (2003) Binding of small molecules to an adaptive protein-protein interface. Proc Natl Acad Sci U S A. 100, (important reference concerning the capture approach of weak binders) 2.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, Kursula, I., Salin, M., Sun, J., Norledge, B.V., Haapalainen, A.M., Sampson, N.S., and Wierenga, R.K. (2004) Understan-ding protein lids: structural analysis of active hinge mutants in triosephosphate isomerase. PEDS (submitted). (about importance of A178L) 4.Norledge, B.V., Lambeir, A.M., Abagyan, R.A., Rottmann, A., Fernandez, A. M., Filimonov, V.V., Peter, M.G., Wierenga, R.K Modelling, mutagenesis and structural studies on the fully conserved phosphate binding loop (loop-8) of triosephosphate isomerase: towards a new substrate specificity. Proteins 42, Schliebs, W. Thanki, N., Eitja, R., Wierenga, R.W. (1996) Active site properties of monomeric triosephosphate isomerase (monoTIM) as deduced from mutational and structural studies. Prot. Science 5, Sun, J., Sampson, N. (1999) Understanding protein lids: kinetic analysis of active hinge mutants in Triosephosphate Isomerase. Biochemistry, 38, 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. Protein Engin. 10(2), References Enzymology of dimer and monomer TIM Wild Type TIM is well understood, and many publications can be found on its catalytic and structural properties. However, some aspects are still poorly understood. We are currently investigating the enzymological properties of wild type TIM (Wt TIM) and monomer TIM (ml1TIM) regarding the loop-6/loop-7 mechanism. Observations in a liganded structure of Leishmania TIM showed P168 (N-terminal hinge) in a planar state which is a strained [2] conformation, which could facilitate loop opening. Additionally a substantial structure adaptation was found in a mutation in the C-terminal hinge. Spherical clashes of the mutant with Leucine at position 178 may be responsible for a forced loop closure [3, 6] (summarized in fig 2). These investigations, and of others [3], are of importance for the engineering part of the project, since only in the closed form the active site is competent for catalysis. This knowledge can then be applied to engineer (more) active enzyme variants regarding poorly binding (new) ligands. Engineering new enzymes Our focus lies on new monomeric TIM enzymes with an altered binding site, but unaltered catalytic properties (fig. 1). This knowledge is based on structures (fig 3.), and obtained by directed evolution and crystallography. Capturing new ligands to new binding pockets of engineered enzymes is challenging, but not impossible [1]. Our approach uses covalently bound suicide inhibitors, and MALDI-TOF mass spectrometry (MALTI-TOF-MS) to find (weak) binding ligands. With NMR we study the dynamic interactions between ligand(s) and our monomeric TIM variants (using 2D-HSQC-NMR). NMR is also used to quantitatively monitor substrate conversion in time (using 1-D NMR) with short time frames in respect to strong interactions, and long time frames for (very) weak interactions (fig 4). Conclusions ►WT TIM and Monomeric TIM are good scaffolds for enzyme engineering ►Wt studies give a useful insight in the catalytic mechanism for future engineering efforts ►Mass Spectrometry and NMR are useful tools for studying: ligand capturing, dynamic ligand-protein interactions, and substrate conversion Figure 3 Connolly surface picture of ml8bTIM V233A-I245A. The active site is at the yellow oval, the new binding pocket is at the green oval. The side chains of Lys13 (catalytic residue) and Lys239 (rim residue) are shown in transparent mode. Regions of negative electrostatic potential are colored red, and positive electrostatic potential are colored blue. In this structure loop- 4 is in the shape of the active site in its active conformation will be different. This picture was made with ICM ( L Figure 1 Overview of the generated variants. MonoTIM, ml1TIM and ml8bTIM have been created in previous studies [5, 7, 4]. All other mutants have been created within the Kealase project. The Wt TIM concerns trypanosomal TIM. Structural information is available from the variants labeled with *. Figure 2 Model of double mutant P168A/A178L based on a 1.06Å structure of AV TIM. In this structure loop 6 and loop 7 are in the ‘open’ conformation. In green are the catalytic residues, in grey the mutation sites in loop 6, and in magenta the expected residues that clash with Leu178. The blue arrows show the hypothesized effect on loop 6 displacement. Loop 6 closure Loop 6 opening clash Loop 6 Loop 7 Trp 170 Ala 168 Leu 178 Glu 167 Lys 13 His 95 Ala 171 Possible interaction Acknowledgements This work is supported by the Academy of Finland (project 53923). The authors would also like to thank Prof. Dr. Koen Augustyns from the University of Antwerp (Belgium) for providing us with Bromohydrogen-acteone phosphate (BHAP). Based on modelling and rational design we are currently investigating several potential ligands that bind the newly engineered binding pocket. Improvement and/or recovery of enzymatic activity will be the focus in directed evolution studies. Figure 4. 1-D NMR spectrum of the decrease of DGAP under the influence of ml1 TIM