PhD projects

ITP FP7 - SBMPs

Structural Biology of Membrane Proteins

PhD project:

Structural characterization of di-myo-inositol-phosphate synthase (DIPPS): the first representative of a new class of enzymes




Principal Investigator:

Margarida Archer, (Membrane Protein Crystallography Group), ITQB, Av. República, EAN, Oeiras, Portugal; archer|itqb.un.l.pt.


In collaboration with:

Helena Santos, (Cell Physiology and NMR Group), ITQB.

Frank Bernhard, Institute of Biophysical Chemistry, Centre of Biomolecular Magnetic Ressonance, J.W. Goethe University of Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany; fbern|bpc.uni-frankfurt.de

The biosynthetic pathway of di-myo-inositol-phosphate (DIP) was elucidated by our team based on the detection of relevant activities in cell extracts of Archaeoglobus fulgidus and the characterization of intermediates and final products by NMR(1). The synthesis of DIP proceeds from inositol-1-phosphate in 3 steps; (i) inositol-1-phosphate is activated to CDP-inositol via the activity of CTP:inositol-1-phosphate cytidylyltransferase; (ii) CDP-inositol is condensed with inositol-1-phosphate to yield a phosphorylated form of DIP (DIP-phosphate); and (iii) finally, DIP-phosphate (DIPP) is dephosphorylated to yield DIP by the action of a phosphatase. We found that the condensation of CTP and inositol-1-phosphate into CDP-inositol, as well as, the synthesis of DIPP from CDP-inositol and inositol-1-phosphare is carried out by a single polypeptide chain (designated as DIPP synthase). The gene was identified, cloned and functionally expressed in E. coli (2). The N-terminal domain of the bifunctional enzyme showed high similarity to the nucleotidyltransferase family, and the C-terminal domain contains the conserved motif (D-G-2[X]-A-R-8[X]-G-3[X]-D-3[X]-D) that is characteristic of the CDP-alcohol phosphatidyltransferase family. The hydropathic profile of the bifunctional enzyme predicts the presence of three transmembrane segments in the C-terminal domain. We cloned and expressed homologous genes from the following hyperthermophiles (optimal growth temperature above 80 ºC): Pyrococcus furiosus, Archaeoglobus fulgidus, Aquifex aeolicus and Thermococcus kodakaraensis. This novel bifunctional enzyme, DIPPS, is the first enzyme reported to use CDP-inositol as a substrate, and should belong to a new class of polyol transferases. Therefore, the determination of its 3D-structure is especially important.


The group headed by Helena Santos is working on the biosynthetic pathways of inositol and glycerol phosphodiesters, which are used for stress adaptation in Archaeoglobus fulgidus. One major effort of her research is to study the thermoadaptation strategies in hyperthermophiles.

The group headed by Margarida Archer aims at the strcutural characterization of this enzymes by X-ray Crystallography (3). The first step involves the crystallization of these membrane-bound proteins using detergents. Crystal growth in lipidic mesophases (in meso) can also be tried.


So far, most structural and functional approaches have been limited by the immense difficulties in the production of sufficient amounts of protein samples in conventional expression systems based on living cells. One goal of the current project is to use the cell-free expression system for the production of the sugar transporters. This work will be performed under the supervision of Frank Bernhard. The open nature of cell-free systems allows the addition of detergents in order to provide an artificial hydrophobic environment for the reaction. This strategy defines a completely new technique for the production of membrane proteins that can directly associate with detergent micelles upon translation. This strategy has already been successfully applied, namely to produce high levels of different G-protein coupled receptors (GPCRs) in an individual cell-free expression system based on Escherichia coli extracts(4).

Reference:

  1. N. Borges, L.G. Gafeira, M.V. Rodrigues, R. Ventura, C. Maycock, P. Lamosa & H. Santos (2006)
    Biosynthetic pathways of inositol and glycerol phosphodiesters used for stress adaptation in Archaeoglobus fulgidus, Journal of Bacteriology, 188, 8128-8135.
  2. M.V. Rodrigues, N. Borges, M. Henriques, P. Lamosa, R. Ventura, C. Fernandes, N. Empadinhas, C. Maycock, M.S. da Costa & H. Santos (2007)
    Bifunctional CTP:inositol-1-Phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase, the key enzyme for di-myo-inositol-phosphate synthesis in several (hyper)thermophiles. Journal of Bacteriology, 189, 5405-5412.
  3. Hickman A.B & Davies D.R. (1997).
    Principles of Macromolecular X-ray Crystallography. In Current Protocols in Protein Science (ed.) pp 17.3.1-15, John Wiley&Sons, Inc.
  4. C. Klammt, D. Schwarz, N. Eifler, A. Engel, J. Piehler, W. Haase, S. Hahn, V. Dötsch, F. Bernhard.
    J Struct Biol., 159, 194-205, (2007)