Projects

1.1. Enzymes involved in the nucleotide metabolism

DHOD PRPPsase ODCase UPRTase UDK CTP_synthase CDA

dCTP deaminase
This enzymes catalyse a reaction very similar to CDA, but it is not zinc dependent. We are studying dCTP deaminase from both Escherichia coli and Methanococcus jannaschii, and have been successful in obtaining good quality crystals of both enzymes.

Dihydropyridimidase
Dihydropyridimidase (DHPase) catalyses the reversible hydrolysis of 5,6-dihydrouracil to N-carbamoyl-beta-alanine, which is the second step in the reductive catabolism of pyrimidine nucleotides. DHPase is homologous with the anabolic enzyme enzyme dihydroorotase. Comparison of the structures of these homologous enzymes may provide valuable insight into the specialisation of catalytic function. We are studying DHPase from both Saccharomyces kluyveri and Dictyostelium discoideum.

RNase PH
In Escherichia coli, the maturation of precursor tRNA molecules is carried out by multiple ribonucleases showing overlapping activities. The exoribonuclease RNase PH is a phosphorylase found to be involved in processing at the 3' end, cleaving the +2 nucleotide following the well characterized tRNA-CCA sequence. A homologous protein has been detected in Bacillus subtilis, which has also been assigned RNase PH. In vitro, RNase PH catalyses the phosphorolysis of polyadenylate and the polymerization of nucleotide diphosphates into a tRNA chain. The phosphorolysis reaction has a requirement of inorganic phosphate and Mg²⁺, whereas the synthetic reaction only requires Mg²⁺. We have determined the 3D structure of B. subtilis RNase PH in a hexameric form and a dimeric form. The hexameric form contain two sulfate ions where one is a marker of the phosphate binding at the active site.

Xanthine phosphoribosyltransferase
Xanthine phosphoribosyltransferase (XPRTase) from Bacillus subtilis is involved in the salvage of purine nucleotides, and catalyses the formation of xanthosine monophosphate (XMP) from xanthine and phosphoribosyldiphosphate (PRPP). XPRTase from B. subtilis shows no sequence similarity to the well-characterised phosphoribosyltransferases, which use both xanthine and other purine bases as substrates, and it is unsual because of its high specificity for xanthine. The structure will provide a basis for a rational kinetic characterisation of e.g. residues essential for catalysis and substrate binding, residues involved in xanthine specificity and the oligomeric state of the active enzyme.

Principal investigators: Sine Larsen , Anders Kadziola and Eva Johansson,

The people currently working on the projects are Susan Arent (DHOD, UPRTase, DHPase, XPRTase), Majbritt Hansen (DHOD), Pernille Harris (ODCase, CTP synthase, UPRTase), Eva Johansson (CDA, dCTP deaminase, PRPPsase, CTP synthase), Anders Kadziola (UPRTase, UCK, RNase PH), Jérôme Le Nours (DHOD) and Jens-Christian Navarro Poulsen (ODCase).

The research on enzymes in nucleotide metabolism involve the following collaborations: Olof Björnberg, University of Lund (dCTP deaminase , DHOD), Karin Hammer, Technical University of Denmark (CTP synthase), Bjarne Hove Jensen, University of Copenhagen (PRPPsase), Kaj Frank Jensen, University of Copenhagen (DHOD, RNase PH, UPRTase, XPRTase), Bjarne Jochimsen, Aarhus University (PRPPsase), Arsalan Kharazmi, Lica Pharmaceuticals (DHOD), Monika Löffler, Philipps-Universität Marburg (DHOD), Jan Martinussen, Technical University of Denmark (CTP synthase, PRPPsase), Jan Neuhard, University of Copenhagen (CTP synthase, CDA , dCTP deaminase , UPRTase, UCK), Jure Piskur, Technical University of Denmark (DHPase), Bent Sigurdskjold, University of Copenhagen (CTP synthase, dCTP deaminase) and Silvia Vincenzetti, University of Camerino (CDA)

Publications

1.2.1. Pectin degrading enzymes

One group of projects has focused on enzymes involved in pectic substance degradation. Most of the work has so far been carried out on enzymes acting on rhamnogalacturonan, the hairy region of pectin. Rhamnogalacturonan I (RG-I) is a complex polysaccharide with a backbone composed of alternating rhamnose and galacturonic acid residues which can be acetylated and methylated. The rhamnose residues of the backbone usually have galactan, arabinan or arabinogalactan attached to C4 as side chains. To enzymatically break down RG-I a complex enzyme system is necessary, mirroring the complexity of the substrate. We have determined the structure of several microbial enzymes taking part in the degradation of RG-I.
The RG-I backbone can be broken down by two main enzymes, Rhamnogalacturonase A (RGase A 1RMG) an endoacting hydrolase cleaving the alpha-1,2 glycosidic between the galacturonic acid and rhamnose residues, and Rhamnogalacturonase B (RGase B, (1NKG), a lyase with novel fold cleaving the alpha-1,4 bond between the rhamnose and galacturonic acid residues, giving an unsaturated product.
Rhamnogalacturonan acetylesterase (RGAE) (1K7C, 1DEO, 1DEX) works in synergy with RGases removing the acetylesters from the backbone.
Beta-1,4-galactanases hydrolyse the glycosidic bonds in galactan and arabinogalactan side chains (see the GH-A section below).

The smooth region of pectin is composed of unbranched polygalacturonan. The galacturonic acid residues can be methylated at the carboxylate group or acetylated at the C2 or C3 position. The backbone of polygalacturonan is broken down by pectin and pectate lyases. Pectate lyase (Pel) from Bacillus agaradherens, Bacillus licheniformis, and Thermotoga maritima have been solved and are being analysed. Complexes of two separate inactive mutants of Thermotoga maritima Pel with a galacturonic acid oligomer of five residues have recently been obtained. These complexes provide added insight into the substrate recognition and catalytic mechanism of Pels. Structural features that confer thermostabilty to the Thermotoga maritima Pel are also being investigated.

Determining the structures is part of our efforts to understand the catalytic mechanism and substrate specificity for these enzymes in collaboration with Novozymes A/S.

Principal investigators: Sine Larsen and Leila Lo Leggio
Other researchers currently involved in the projects: Jens-Christian Navarro Poulsen, Annette Langkilde, Malene Hillerup Jensen and Ditte Welner.

1.2.2. Enzymes in clan GH-A

Clan GH-A is a superfamily of glycoside hydrolases sharing a conserved 8-fold beta/alpha-barrel architecture, and a conserved retaining mechanism but having different substrate specificities. We are particularly interested in relating structural differences to polysaccharide specificities.
Over the years we have determined the structures of enzymes belonging to GH5 (1GZJ) and GH10 (1K6A, 1GOK, 1GOM, 1GOQ, 1GOR, 1GOO and 1E5N).
A recent project has elucidated the structural basis of thermostability, pH optimum and substrate specifity in GH53 galactanases (Ryttersgaard et al, 2002; Le Nours et al., 20 03; Ryttersgaard et al., 2004, 1FHL, 1FOB, 1HJS, 1HJU, 1HJQ, 1R8L, 1UR0, 1UR4) in collaboration with Novozymes A/S.
A collaboration with Henrik Stålbrand and Lars Anderson at the University of Lund has led to the determination of the structure of the GH26 beta-1,4-mannanase from C. fimi, revealing an unexpected Ig-like domain associated with the catalytic domain. (Le Nours et al, 2005).

Publications

Transcription factors are generally DNA binding proteins involved in regulation of gene expression. In collaboration with Karen Skriver and Addie Nina Olsen at the Institute of Molecular Biology, University of Copenhagen, we have determined the first structure of a NAC domain dimer (1UT4, 1UT7) The NAC domain is the conserved DNA-binding domain of NAC proteins, plant specific transcription factors involved in plant development and response to stress.

Principal investigator: Leila Lo Leggio
Other researchers currently involved in the projects: Heidi A. Ernst, Azhar Mahmood Chaudry and Lotta Helgstrand.

The electrostatic potential mapped onto the molecular surface with direction of the dipole moment shown (green arrows). Blue represents positive charge distribution and red negative charge. The view is along the pseudo-twofold axis: front view (left) and back view (right). [3]

1.4.1. Heme containing enzymes

Structural studies of heme containing enzymes were among our first protein crystallographic projects and have led to the determination of the three dimensional structure of the peroxidase from the fungus Coprinus cinereus (1H3J, 1LY8, 1LY9, 1LYC, 1LYK) and the cytochrome c4 from Pseudomonas stutzeri (1ETP). The study of the former enzyme is part of a collaborative project with Novozymes A/S, and the latter protein with Prof. Jens Ulstrup's group from the Technical University. Presently we focus on structure determinations for the reduced and oxidised forms of the cytochrome c4. The work on the Coprinus peroxidase has primarily been conducted by Pernille Harris and Karen Houborg, and the work on the cytochrome c4 by Anders Kadziola, Pernille Harris and Allan Nørgaard.

Our investigations in this area comprise studies of cytidine deaminase (see 1.1.) and carboxypeptidase A. The latter enzyme was one of the first enzymes with a known three dimensional structure and a models for its catalytic function was proposed at an early stage. Based on the data from a new crystal form and for the Cd substituted enzyme at different pH and chloride concentrations we hav been able to present a revised reaction model for this enzyme. [4, 5] (1CPX, 1ELM, 1EE3, 1ELL) The people who have been involved in these studies are Anders Kadziola, Jens Bukrinsky and Anette Frost Jensen.
Principal investigator: Sine Larsen

Publications

Cathepsin C
Cathepsin C or dipeptidyl peptidase I (DPPI) is a peptidase with a large prodomain and a complex activation reaction. The structure of the DPPI from rat was determined by MIR, it revealed a unique tetrameric structure where the residual prodomain adopts an essential role in the active site [7] (1JQP, patent). The research is continued by investigatiions of inhibitor complexes of the human enzyme. Collaborators: Unizyme.

Kinases involved in signal transduction
The growth factor activated AGC kinases constitute a major subgroup of the serine/threonine protein kinases. They contain an essential serine/threonine phosphorylation site in a hydrophobic motif, which is C-terminal to their kinase domain. The interaction between the hydrophobic motif and the hydrophobic pocket in PDK1 has been elucidated by modelling and docking studies, and two basic residues were identified to be important for phosphate binding. Collaborators: Morten Frödin and Steen Gammeltoft, Glostrup Hospital.

The p38 mitogen activated protein (MAP) kinase belongs to the CMGC subgroup of the serine/threonine protein kinases. It is part of the stress activated transduction cascade responsible for cytokine production. Several structures of the p38 MAP kinases are known and they have been used to design inhibitors. We are optimising the crystallisation conditions for the native human p38 MAP kinase in order to be able to investigate the protein inhibitor complexes. Collaborators: Morten Dahl Sørensen and Frederik Björkling, Leo Pharmaceutical Products.
Principal investigator: Sine Larsen

Publications

The interactions between chiral molecules are important for most biological processes. The enantiomers of a chiral molecule would frequently lead to different biological functions and so it is important to use the pure enantiomers as drugs, herbicides and pesticides. Most of the synthetic routes that are used in their preparation do not lead to pure enantiomers but to a racemic mixture. Therefore it has become increasingly important to understand the physico-chemical and structural background for the processes that are used to isolate the pure enantiomers. We have approached this by studying two principally different types of problems. One where we investigate the thermodynamic and structural aspects of the crystallisation of racemates, and in the other where we study the resolution of racemates through diastereomeric salt formation.

These investigations constituted Heidi A. Ernst's cand. scient. studies and part of Katalin Marthi's Ph.D. project. At present Ph.D. student Henning O. Sørensen, cand. scient. student Anne Egebjerg and cand. scient. student Erik Riis are involved in these studies.
Principal investigator: Sine Larsen

Publications

We use the information that the X-ray diffraction data contain about the thermally averaged electron density to study interatomic interactions in crystals. These investigations require very accurate high resolution X-ray diffraction data. Frequently a complementary neutron diffraction study is performed to get accurate parameters for the hydrogen atoms in the crystals. The collaboration with professor Robert F. Stewart, Carnegie-Mellon University, plays an important role for these investigations, which also involve a significant amount of program development for the program system VALRAY, which is primarily used for the computations involving the diffraction data. The topological analysis developed by R. Bader for theoretical electron densities is successfully employed to study the properties of the experimental electron densities. It has been natural to complement these investigations with comparative quantum chemical calculations both on the molecular entities and the crystal. For the crystals we primarily use periodic Hartree-Fock calculations (CRYSTAL98). The investigations also encompass methodological research on the treatment of hydrogen atoms in charge density studies based solely on X-ray diffraction data and analysis of the parameters for data collection with a CCD detector.

The systems studied are molecular crystals that display interesting interatomic interactions. An example is the recently completed research on very short hydrogen bonds conducted by Ph.D. Claus Flensburg, Ph.D. Dennis Madsen and cand. scient. Annette Langkilde. We are currently exploiting the potential of the methods to include acentric crystals and are extending the work to the study of weaker intermolecular interactions. These investigations are carried out by Ph.D. student Henning O. Sørensen, cand. scient. Anders Ø. Madsen, cand. scient. Jette Oddershede and cand. scient. student Erik Riis.

Last modified: Mon 26. Sept 2005