Structural enzymology: a quantitative approach

Contact details:

Professor Rik Wierenga
Biocenter Oulu and Department of Biochemistry, University of Oulu
P.O.Box 3000, FIN-90014 University of Oulu, Finland
Phone: +358-8-553 1199, Fax: +358-8-553 1141
E-Mail: firstname.lastname@oulu.fi

Introduction

We study, at the molecular level, all aspects of enzyme-ligand interactions. Our research is aimed at a better understanding of how enzymes achieve their remarkable biocatalytic properties. How do enzymes really work? (Blow, 2000). Our principal expertise concerns protein crystallographic structure determinations. This expertise is complemented by an extensive set of complementary techniques, ranging from enzymological methods, spectroscopic studies, and directed evolution approaches to biocomputing, bioinformatics and chemistry. This complementary expertise is available either in-house or through collaborations.

Research interests

Enzymes are proteins with catalytic properties; they are the products of natural evolution. Enzymes have evolved to catalyse a wide range of reactions with high substrate and reaction selectivity. Both the affinity as well as the catalytic efficiency of enzymes have evolved such as to be optimal for the metabolic requirements of the organism. Consequently most enzymes operate at room temperature, at ambient pressure and under conditions of neutral pH. The rate enhancements achieved by enzymes are enormous, typically in the range of 10⁵ to 10⁸ (Koeller and Wong, 2001), but in extreme cases of the order of 10¹⁷ (Radzicka and Wolfenden, 1995; Wolfenden, 1999). From the studies of Wolfenden and others it emerges that enzymes have evolved to bind the high energy transition state much more strongly than the substrate or the product, thus allowing for catalysis to happen. Achieving the detailed understanding of the reaction mechanisms of enzymes is an intellectual challenge. It is also of crucial importance for being able to harness the enormous catalytic power of enzymes for the synthesis of taylor-made compounds in lab scale organic chemistry and large scale industrial processes (Koeller and Wong, 2001). This is particularly important because many naturally evolved enzymes have a substrate and reaction specificity which is different from the reactions of practical importance and consequently wild type enzymes have to be engineered such as to optimize the required substrate and reaction selectivity. Furthermore, precise understanding of the reaction mechanism facilitates the discovery of very tight binding transition state analogues for use as enzyme inhibitors, which are for example potential drugs in medical applications (Schramm, 2005). Principally, three classes of enzymes are being studied, which are triosephosphate isomerases (TIM), CoA-dependent enzymes and prolyl-4-hydroxylases (Figure 1).

Research infrastructure

The principle technique used by us is the method of protein crystallographic structure determinations. Our research group is responsible for maintaining the infrastructure of the protein crystallographic set up at the Department of Biochemistry (Figure 2). Our expertise on protein crystallographic methods is complemented by enzymological and biocomputational approaches, either in-house or via collaborations. The Department of Biochemistry is well equipped for carrying out molecular biological mutagenesis and protein purification work. In addition steady state enzyme kinetic studies and affinity measurements, for example by Biacore or calorimetry are done regularly. Also protein stability studies, for example by making CD-melting curves are routinely carried out. Our studies of the natural wild type enzymes complexed with the natural substrates and ligands are complemented by studying the properties of mutated variants complexed with modified ligands. Molecular biological methods are used to make the mutated variants and organic chemistry synthetic approaches are used for making the modified ligand. The organic chemistry synthesis is done either in house or via collaborations. Chemistry is an essential part of our research, both for the synthesis of special compounds, as well as for discussions on the chemical aspects of the reaction mechanisms.

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Research projects

Triosephosphate isomerases and the engineering of non-natural enzymes.

Figure 3.Results of an ICM docking calculation, using the closed A-TIM structure for docking of compound B123. This compound is a substrate analogue; it has been synthesised by Matti Vaaisma of the group of Marja Lajunen, Chemistry Department. The omega-end of this compound is docked into a pocket near Ile245. The sulfonate moiety is docked near the corresponding phosphate position of wildtype TIM, visualised by the superimposed structure of the transitionstate analogue phosphoglycolohydroxamate.

In this project we study the properties of the wild type enzyme, as well as of monomeric variants. The latter variants have been obtained by mutating the loops which in wild type stabilise the dimer interface. The studies of the monomeric variants are aimed at changing its substrate specificity. This work is done in collaboration with the Chemistry department (Lajunen, Mattila) and the Process Engineering department (Neubauer, Ylianttila).

The wild type TIM studies concern atomic resolution studies of leishmania TIM, complexed with transition state analogues such as 2-phosphoglycollate and phosphoglycolohydroxamate. These crystals diffract to approximately 0.82 Å resolution. The refinement of the structures at this resolution provides information about the geometry at a unique precision and detail. Therefore, such structures provide information on the protonation state of the active site, which is important for the understanding of the reaction mechanism.

The work on monomeric TIM builds on the characterisation of ml8bTIM (Norledge et al, 2001) and A-TIM (Alahuhta et al, 2008). A-TIM has been shown to have favourable binding properties which are now being explored in directed evolution experiments aimed at introducing catalytic activity. Such a variant will be referred to as a kealase, being able to convert an alfa-hydroxy-ketone into a chiral alfa-hydroxy-aldehyde.

CoA-dependent enzymes

We study the catalytic properties of four different CoA-dependent enzymes: thiolases, racemases, enoyl-CoA isomerases, and thioesterases. Each of these enzymes catalyses a thioester dependent reaction of crucial importance in lipid metabolism. Thioesters are more reactive than oxygen esters and lipid molecules are usually stored as oxygen esters whereas lipid metabolism is thioester dependent. The higher reactivity of the thioesters (Figure 4) is caused by the larger atomic radius of a sulphur atom as compared to an oxygen atom, thereby causing differences in the preferred resonance structures.

The studies of each of these enzymes are collaborative efforts with foreign and finnish research groups, in particular with the groups of Hiltunen (University of Oulu) and Pihko (University of Jyväskylä). The work on thiolase covers a range of different thiolases, both bacterial and human. The work on the bacterial thiolase concerns the tetrameric Zoogloea ramigera thiolase, which structure is known and of which several mutants have been made. Six human thiolase isozymes have been identified: one cytosolic thiolase, three mitochondrial, and two peroxisomal thiolases. For each of these thiolases we aim to characterise the enzymological and structural properties. In addition we have started collaborative efforts to obtain transition state analogues.

 The work on the transition state analogues of thiolase is not only relevant for better understanding of its reaction mechanism, but is also done in the context of finding inhibitors for parasitic thiolases (Schramm, 2005). This research and the research aimed at developing new catalytic activities on the A-TIM framework using directed evolution approaches (Tracewell and Arnold, 2009) also fit in our continuous efforts to actively support the development of a regional structure-based-biotech industry (http://www.strucbiocat.oulu.fi/).

Prolyl-4-hydroxylases

A prolyl-4-hydroxylase hydroxylates prolines at the 4-position, using molecular oxygen as its substrate and alfa-ketoglutarate as its cofactor. The product is the trans-C4-hydroxyl enantiomer (Figure 5). The enzyme uses also non-heme iron as a cofactor. The P4H's belong to a superfamily of enzymes which have as a core the jelly-roll fold, which is also referred to as the double stranded beta-helix (DSBH) -fold, because it is comprised of 8 beta-strands. In the active enzyme the iron is in its Fe2+ state, liganded to side chains of two histidines and an aspartate.

In collaboration with the Myllyharju group (University of Oulu) we have solved in 2006 the crystal structure of the monomeric chlamydomonas P4H in its apo form, with no bound Fe²⁺ and no bound ligand or cofactor. More recently a crystal form of this P4H has been obtained with Zn²⁺ and an alfa-ketoglutarate analogue bound in the active site. Also we have now established the mode of binding of a substrate peptide. Much efforts are now aimed at the characterisation and crystallisation of the tetrameric alfa2-beta2 complex of human P4H. In this complex the alfa-chain contains the catalytic domain (at its C-terminus), whereas its N-terminal domain is known to be involved in peptide binding. The beta-chain is PDI, which is a key component of the folding machinery in the ER; structural studies on this subunit are being carried out with the Ruddock group (University of Oulu). The P4H-alfa-chain is insoluble without the beta-subunit. Nothing is known about the mode of interaction of the PDI-beta-subunits with the alfa-subunits. In parallel with the crystallisation efforts on the full length P4H we are crystallising its domains and the crystal structure of the proline binding domain of the alfa-chain has been solved already.

Major challenges

The above projects have several major challenges. For example, can we make a non-natural enzyme with a taylor-made kealase catalytic activity? Can we get a better understanding of the role of waters in the active site for the catalytic power of enzymes? Can we crystalise and solve the crystal structures of the multi-domain and multi-subunit assemblies, such as MFE-1, the human P4H tetramer and the human, mitochondrial alfa2-beta2-dimer beta-oxidation complex? Each of these goals can only be achieved through collaborations and will require innovative, multidisciplinary approaches.

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Research Group Members

Project Leader:
Rik Wierenga

Senior and Post doctoral Investigators:
Antti Haapalainen
Kristian Koski
Tiila Kiema
Rajaram Venkatesan
Vanja Kapetaniou

Ph.D. Students:
Prasad Kasaragod
Jothi Anantharajan
Rajesh Harijan
Goodluck Onwukwe

Pro Gradu Students:
Howard Lee

Laboratory Technician:
Ville Ratas

Recent Publications

Venkatesan, R., Alahuhta, M., Pihko, P.M. and Wierenga, R.K. (2011) High resolution crystal structures of triosephosphate isomerase (TIM) complexed with its suicide inhibitors: the conformational flexibility of the catalytic glutamate in its closed, liganded active site. Prot Sci, (in press).

Janardan, N., Paul, A., Harijan, R. K., Wierenga, R. K. and Murthy, M.R.N. (2011) Cloning, expression, purification and preliminary X-ray diffraction studies of a putative Mycobacterium smegmatis thiolase. Acta Cryst F, (in press).

Papai, I., Hamza, A., Pihko, P.M., Wierenga R.K. (2011) Stereoelectronic requirements for optimal hydrogen bond catalyzed enolization. Chem Eur J, 17, 2859-2866.

Salin, M., Kapetaniou, E.G., Vaismaa, M., Lajunen, M., Casteleijn, M.G., Neubauer, P., Salmon, L. and Wierenga, R.K. (2010) Crystallographic binding studies with an engineered monomeric variant of triosephosphate isomerase. Acta Cryst, D66, 934-944.

Wierenga, R.K., Kapetaniou, E.G. and Venkatesan, R. (2010) Triosephosphate isomerase: a highly evolved biocatalyst. Cell Mol Life Sci, 67, 3961-3982.

Kasaragod, P., Venkatesan, R., Kiema, T.R., Hiltunen, J.K. and Wierenga, R.K. (2010) The crystal structure of liganded rat peroxisomal multifunctional enzyme type 1: a flexible molecule with two interconnected active sites. J Biol Chem, 285, 24089-24098.

Alahuhta, M. and Wierenga R.K. (2010) Atomic resolution crystallography of a complex of triosephosphate isomerase with a reaction intermediate analogue: new insight in the proton transfer reaction mechanism. Proteins, 78, 1878-1888.

Hiltunen, J.K., Chen, Z., Haapalainen, A.M., Wierenga, R.K. and Kastaniotis, A.J. (2010) Mitochondrial fatty acid synthesis - an adopted set of enzymes making a pathway of major importance for the cellular metabolism. Progress in Lipid Research, 49, 27-45.

Meriläinen, G., Poikela, V., Kursula, P. and Wierenga, R.K. (2009) Thiolase reaction mechanism studies: the importance of Asn316 and His348 for stabilizing the enolate oxyanion intermediate in the Claisen condensation reaction. Biochemistry, 48, 11011-11025.

Koski, M.K., Hieta R., Hirsilä, M., Rönkä, H., Myllyharju, J. and Wierenga, R.K. (2009) The structure of an algal prolyl 4-hydroxylase complexed with a proline-rich peptide reveals a novel buried tripeptide binding motif. J Biol Chem, 248, 25290-25301.

Chen, Z., Kastaniotis, A.J., Miinalainen, I.J., Venkatesan, R., Wierenga, R.K. and Hiltunen, J.K. (2009) 17beta-hydroxysteroid dehydrogenase type 8 and carbonyl reductase type 4 assemble as a ketoacyl reductase of human mitochondrial FAS. FASEB J, 23, 3682-3691.

Pihko, P. Rapakko, S. and Wierenga, R.K. (2009) Oxyanion holes and their mimics. In “Hydrogen Bonding in Organic Synthesis”, Editor: Pihko, P., Wiley-VCH Verlag, Weinheim, Germany, pp 43-71.

Donnini, S., Villa, A., Groenhof, G., Wierenga, R.K., Mark, A.E. and Juffer, A.H. (2009) Inclusion of ionization states of ligand in affinity calculations. Proteins, 76, 138-15

Meriläinen, G., Schmitz, W., Wierenga, R.K., and Kursula, P. (2008) The sulfur atoms of the substrate CoA and the catalytic cysteine are required for a productive mode of substrate binding in bacterial biosynthetic thiolase, a thioester-dependent enzyme, FEBS Journal, 275, 6136-6148

Nguyen, V.D., Wallis, K., Howard, M.J., Haapalainen, A.M., Salo, K.E.H., Saaranen, M.J., Sidhu, A., Wierenga, R.K., Freedman, R.B., Ruddock, L.W., and Williamson, R.A. (2008) Alternative conformations of the x region of human protein disulphide-isomerase modulate exposure of the substrate-binding b' domain. J Mol Biol, 383, 1144-1155

Chen, Z., Pudas, R., Sharma, S., Smart, O.S., Juffer, A.H., Hiltunen, J.K., Rik K. Wierenga, R.K., Haapalainen, A.M. (2008) Structural Enzymological studies of 2-Enoyl-Thioester Reductase of the Human Mitochondrial FAS II pathway: New Insights into Its Substrate Recognition Properties. J Mol Biol, 379, 830-844

Alahuhta, M., Salin, M., Casteleijn, M.G., Kemmer, K., El-Sayed, I., Augustyns, K., Neubauer, P., Wierenga, R.K. (2008) Structure-based protein engineering efforts with a monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties. PEDS, 21, 257-266

Alahuhta, M., Casteleijn, M.G., Neubauer, P., Wierenga, R.K. (2008) The A178L mutation in the C-terminal hinge of the catalytic loop-6 of triosephosphate isomerase (TIM) induces a closed-like conformation in dimeric and monomeric TIM, Acta Cryst, D64, 178-188

Koski, M.K., Hieta, R., Böer, C., Kivirikko, K.I., Myllyharju, J., and Wierenga, R.K. (2007) The active site of an algal prolyl 4-hydroxylase has a large structural plasticity. J Biol Chem, 282, 37112-37123

Haapalainen, A.M., Meriläinen, G., Pirilä, P.L., Kondo, N., Fukao, T., Wierenga, R.K. (2007) Crystallographic and kinetic studies of human mitochondrial acetoacetyl-CoA thiolase (T2): the importance of potassium and chloride ions for its structure and function. Biochemistry, 46, 4305-4321

Bhaumik, P., Schmitz, W., Hassinen, A., Hiltunen, J.K., Conzelmann, E., Wierenga, R.K. (2007) The 1,1-proton transfer reaction mechanism by a-methyl-acyl-CoA racemase is catalysed by an aspartate/histidine pair and involves a smooth, methionine-rich surface for binding the fatty acyl moiety. J Mol Biol, 367, 1145-1161

Sakurai, S., Fukao, T., Haapalainen, A. M., Zhang, G., Yamada, K., Lilliu, F., Yano, S., Robinson, P., Gibson, M. K., Wanders, R.J., Mitchell, G.A., Wierenga, R.K., Kondo, N. (2007) Kinetic and Expression Analyses of Seven Novel Mutations in Mitochondrial Acetoacetyl-CoA Thiolase (T2): Identification of a Km Mutant and an Analysis of the Mutational Sites in the Structure. Molecular Genetics and Metabolism, 2007, 370-378

Taskinen, J.P., van Aalten, D.M., Knudsen, J. and Wierenga, R.K (2007) High resolution crystal structures of unliganded and liganded human L-ACBP reveal a new mode of binding for the acyl-CoA ligand. PROTEINS: Structure, Function, and Bioinformatics, 66, 229-238

Casteleijn, M.G., Alahuhta, M., Groebel, K., El-Sayed, I., Augustyns, K., Lambeir, A.M., Neubauer, P., Wierenga, R.K. (2006) Functional role of the conserved active site proline of triosephosphate isomerase. Biochemistry, 45, 15483-15494

Taskinen, J.P., Kiema T.R., Hiltunen J.K. and Wierenga, R.K. (2006) Structural studies of MFE-1: the 1.9Åcrystal structure of the dehydrogenase part of rat peroxisomal MFE-1. J. Mol. Biol., 355, 734-746

Haapalainen, A.M., Meriläinen G. and Wierenga, R.K. (2006) The thiolase superfamily: condensing enzymes with a common structural framework but very diverse catalytic properties. TiBS, 31, 64-71

Donnini, S., Groenhof, G., Wierenga, R.K., Juffer, A.J. (2006) The planar conformation of a strained proline ring: a QM/MM study. PROTEINS: Structure, Function, and Bioinformatics, 64, 700-710

References

Alahuhta, M., Salin, M., Casteleijn, M.G., Kemmer, K., El-Sayed, I., Augustyns, K., Neubauer, P., Wierenga, R.K. (2008) PEDS, 21, 257-266.

Blow, D. (2000) Structure, 8, R77-81.

Koeller, K.M., and Wong, C-H. (2001) Nature, 409, 232-240.

Norledge BV, Lambeir AM, Abagyan RA, Rottmann A, Fernandez AM, Filimonov VV, Peter MG, Wierenga RK. (2001) Proteins, 42, 383-389.

Radzicka, A. and Wolfenden, R. (1995) Science, 267, 90-993.

Schramm, V.L. (2005) Current Opinion in Structural Biology, 15, 604-613.

Tracewell, C.A., and Arnold, F.H. (2009) Current Opinion in Chemical Biology,13, 3-9.

Wolfenden, R. (1999) Bioorganic and Medicinal Chemistry, 7, 647-652.

Positions available

Please contact Rik Wierenga for the availability of open positions for Ph.D. students and postdocs. Such positions concern the research topics described above as well as other projects.