1. Towards new enzymes: From Triosephosphate Isomerase to Kealases Peter Neubauer University of Oulu 31.08.2006 (PDB2006)

2. Peter Neubauer Introduction Norledge et al; Proteins: structure, function and genetics (2001) Enzyme of the glycolysis pathway Wild type enzyme is a dimer that consists of two identical (  )8-fold subunits with the size of 250 residues Loop-1 and 4 (subunit 1) and loop-3 (subunit 2) form the dimer interfase Highly specific binding pocket (phosphate) is formed by loops 6, 7 and 8 Catalyses the inter- conversion of dihydroxy acetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (DGAP)

3. Loop6 Loop7 Peter Neubauer Introduction Catalytic residues: Lysine 13 Histidine 95 Glutamate 167 Ligand is represented as green density Loop-6 closes upon ligand binding Loop6 Loop7 Catalytic residues: Lysine 13 Histidine 95 Glutamate 167 Ligand is represented as green density Loop-6 closes upon ligand binding Reaction mechanism as proposed by Kursula et al. Eur. J. Biochem (2001)

4. Peter Neubauer Dimer to monomer Borchert et al (1993; 1994) Thanki et al. (1997) e.g. Wierenga (2001) ml8b TIM Norledge etl. (2001) Dimeric stabilization is important for the catalytic machinery in wild type TIM MonoTIM and ml1 TIM are active enzymes and catalyze the substrates in a similar fashion The affinity of the monomeric TIMs for the transition state analogues is lower The turnover number for the monomeric TIMs is lower Ml8b TIM does no longer convert any known substrates. However the active site remains the same.

5. Peter Neubauer Monomeric TIM activity Mode of binding of 2PG in wild type and monomeric TIM (ml1 TIM). Lys 13 2PG Glu 167 His 95 Loop-6 The turnover number is 1000 lower in monomeric TIM compared to wild type This is correlated with a different environment, such as an increased solvent accessibility The reduction in affinity for 2PG is less than for the phosphate moiety Lysine 13 and Histidine 95 show more conformational flexibility compared to wild type. Monomeric TIMs are more “floppy“ The more accessible active site is an interesting starting point for enzyme engineering

6. Peter Neubauer Tools for new enzymes What? Design of new artificial enzymes Design and organic synthesis of new substrates Creating active enzymes Chemistry Wild type studies iterative process (Random) mutagenesis Selection of best mutants Screen for activity Pool of enzymes TIM (variants) [inactive] Kealases InputOutput Mass Spectrometry X-Ray crystallography NMR Multidisciplinary approach of the biocatalyst consortium Importance of chirally active aldehydes Our Start:Our goal: Wild type studies

7. Peter Neubauer Recent wild type studies Investigations on the properties of the conserved active site proline identified a molecular switch to bring the enzyme in a competent state for catalysis upon ligand binding. Enzymology studies on P168A mutant versus wild type show a dramatic drop in K cat /K m The affinity for 2PG is reduced The mode of binding of 2PG in wild type differs from P168A mutant. Conclusions: ►Proline 168 is needed for conformational strain around the catalytic glutamate. This strain is transferred to the catalytic glutamate. ►Ligand binding triggers the conformational switch of the catalytic machinery needed for catalysis. Poster: ”The functional role of the conserved active site proline of triosephosphate isomerase”

8. Peter Neubauer Why monomeric TIM? L Monomeric TIM is a very suitable protein for biocatalysis: small size (suitable for NMR; easy to crystallize): so far all constructs of monomeric TIMs could be crystallised) easily expressed in high amounts of soluble protein in E.coli Monomeric protein has advantages (Vanvaca et al. PNAS 2004) No cofactors needed In the closed conformation the binding site is an extended groove: ml8b TIM (*) Its wild type precursor is the extensively studied TIM.

9. Peter Neubauer Tailored ligands The Concept Catalytic head group stays the same Achor part contributes most to the binding affinity The anchor is optimized for maximum affinity, but can be cleavable A possible Substrate A Possible Inhibitor (cleavable) AnchorCatalytic head group Original binding site CatalyticCatalytic - S i t e Extended binding pocket Enzyme surface

10. Peter Neubauer A-TIM Over 20 constructs based on ml8b TIM have been created Several mutations have been investigated: pocket, rim, active site and loop mutants. Many have structures have been solved A variant with a deeper and wider binding groove have been created: A-TIM A-TIM is as stable as ml1 TIM in solution The active site is identical to wild type TIM Active site Extended groove

11. Peter Neubauer A-TIM Over 35 novel tailored ligands have been created Binding studies with NMR, Surface Plasma Resonance and X-ray crystallography have identified lead compounds which can be called “binders” Site directed evolution is the next logical step to convert “binders“ to substrates Site directed evolution has worked previously to improve activity If A-TIM variants converts α-hydroxy ketones to α-hydroxy aldehydes, they can be called Kealases Saab-Rincon et al. Protein eng. (2001): Poster: Non-natural enzymes: Directed evolution of a monomeric triosephosphate isomerase variant – a case study

12. Peter Neubauer Recent findings In wild type Proline 168 acts as a molecular switch upon ligand binding. Manuscript is submitted A study on C-terminal hinge residue A178 in wild type and monomeric TIM provides interesting details about the “floppy“ nature of monomeric TIM. Manuscript is under preparation An atomic resolution structure of wild type TIM with PGH (0.82 Å) will give new insights in the reaction mechanism (data under investigation) 1.06 Å detail of A-TIM variant

13. Peter Neubauer Recent findings In X-ray structures the three following compounds are seen as “binders“ In an X-Ray structure BHAP (bromo hydroxyactone phosphate; a suicide inhibitor) has been found to react in the active site similar to wild type. This finding indicates that A-TIM can still convert the original substrate (K cat is too low to measure): A-TIM is an active molecule Citric acid Malic acid MW-1 Mare, de La et al. Biochem J (1972)

14. Peter Neubauer New A-TIM enzymes towards the transformation of new modified nucleosides New result: citric acid binds to the active site of A-TIM (structure 1.5 Å) Also malic acid and compound MV-1 bind to the active site (seen in X-ray) Surface plasma resonance method to screen for binders has been established (Dr. Päivi Pirila) Citric acid as positive control binds in the assay specifically Other compounds tested (with good results): A possible application for A-TIM Citric acid Malic acid MW-1

15. Peter Neubauer Acknowledgments Faculty of Science Department of Biochemistry Prof. Rik Wierenga Markus Alahuhta Mikko Salin Ville Ratas Department of Chemistry Prof. Jouni Pursiainen Ritva Juvani Prof. Marja Lajunen Matti Vaismaa Dr. Sampo Mattila Nanna Alho Faculty of Technology Bioprocess Engineering Prof. Peter Neubauer Dr. Mari Ylianttila Marco Casteleijn Lilja Kosamo This work has been supported by the Academy of Finland (project 53923) and Tekes (project Biocatnuc) website: http://www.oulu.fi/bioprocess/http://www.oulu.fi/bioprocess/ Collaborators University of Kuopio Department of Pharmaceutical Chemistry Prof. Seppo Lapinjoki Technical University of Helsinki Dr. Petri Pihko University of Antwerp, Belgium Prof. Koen Augustyns Prof. Anne-Marie Lambeir