Creating new enzymesDirected Evolution of Triosephosphate Isomerase Mirja Krause1, Marco G. Casteleijn1, Markus Alahuhta2, Mikko Salin2, Ville Ratas2, Rik Wierenga2, Peter Neubauer1 1 Bioprocess Engineering Laboratory, 2Biochemistry - Oulu University and Biocenter Oulu INTRODUCTION Triosephosphate isomerase (TIM) is a glycolytic enzyme with very high substrate specificity (fig 1). The wild type enzyme is a dimer, and each subunit has the classical TIM-barrel fold [1]. Several loop modifications resulted in a monomeric enzyme (ml1TIM) [2] which remains active, although less so than the wild-type. Subsequently this has resulted in a new variant (A-TIM) with a much more extensive binding [3, 4]. Our current studies attempt to change the substrate specificity of monomeric TIM. important a single point mutation for the generation of a competent active site is [4, 5, 6]. Ala Loop 6 Ser r Loop 4 Loop 8 Ile Figure 2. TIM-barrel fold of A-TIM (grey). Starting points are shown in black (A178L loop 6, S96P loop 4, I245A loop 8). RESULTS Figure 1. Wild type TIM (Wt-TIM) has high substrate specificity, and only catalyses the interconversion of the α-hydroxykentone DHAP and the α-hydroxyaldehyde D-GAP. The transition state analogue, 2-phosphoglycolate (2-PG), can be seen on the right. METHODS Directed Evolution The substrate binding loops of ATIM were modified by site-directed mutagenesis to obtain different mutant enzymes as a starting point for directed evolution (fig. 2). In former studies it has been shown how Loo p7 va a l al s gl a Loo gln y ly p8 u s Loo p4 Figure 3. TIM-barrel fold of A-TIM (grey). Targeted areas for directed evolution are located in loop 4, loop 7 and loop 8 Figure 4. Flowchart showing an overview of the ATIM library creation, resulting in 16 different libraries Directed evolution was performed in a fully random approach (epPCR) and a targeted approach. The extensive structural knowledge in this project [1, 4, 5, 6] has been the basis for the choice of amino acids that were targeted through Megaprimer PCR [7]. Library creation Our aim is to build a platform of TIM variants (Kealases) with a widened substrate range. Therefore we established by rational design a library of A-TIM variants (fig. 4). CONCLUSIONS The created libraries are used to screen in vivo for different ATIM mutants which can take over the reaction originally catalyzed by other enzymes or catalyze novel reactions. REFERENCES 1. Kursula, I., and Wierenga, R.K. (2003). J. Biol. Chem 278, 9544-9551 2. Thanki, N., et al, (1997). Protein Engin. 10(2), 159167 3. Norledge, B.V., et al, (2001) Proteins 42, 383-389. 4. Alahutha M, et al. (2008) PEDS Advance Access published on Jan 31. 5. Casteleijn et al. (2006) Biochemistry 6. Alahutha et al. (2008) Acta D. 7. Wu Wu et al. (2005) Nucleic Acids Res. 33:e1107. ACKNOWLEDGEMENTS This work is supported by the Academy of Finland (project 53923 & 11784) More information: www.oulu.fi/bioprocess/biocatalysis [Kealases and Biocat projects]