Tesis doctoral de Fernando Oscar Garcés Ferreira
Escherichia coli, an enterobacteria that inhabit the animal gut, can use the l-ascorbate as carbon source. The transport of l-ascorbate to the intracellular environment and its transformation into d-xylulose-5-p, an intermediate which is subsequently metabolized by the pentose phosphate pathway, is mediated by enzymes encoded by the ula regulon (utilization of l-ascorbate). the ula system is formed by two transcriptional units: ulag cistron and ulaabcdef operon. The gene ulag encodes a putative l-ascorbate-6-phosphate lactonase and ulaabcdef encode the three enzymes of the phosphotransferase system (ulaabc) and the three catabolic enzymes (uladef). The transcription of ula genes is regulated by the ular transcription factor, member of the deor eubacterial repressors family. In the absence of l-ascorbate, as the carbon source in the medium, ular protein maintains the ula regulon negatively repressed. The availability of l-ascorbate in the growth medium releases ular-mediated repression and thereby activates the transcription of genes encoding all enzymes required for l-ascorbate metabolism. although several reports help to understand the complexity of this regulatory mechanism some main points remain elusive to date. For instance, the molecular details and the structural changes that take place in the mechanism of ular repression and activation by l-ascorbate are not known. in addition, the function of the ulag encoded protein, classified as a putative l-ascorbate-6-phosphate lactonase, has not been experimentally proven. the aim of this project is to bring new insights on ula system regulation and function. Thereby, in this work the crystallization of ular and ulag proteins were approached in order to obtain their three-dimensional structures. These data should provide a deep knowledge about ular regulation mechanism on ula system and the role that ulag plays in the metabolism of l-ascorbate. in the present work ular gene was cloned and its product (ular protein) was expressed and purified to obtain a soluble, functional and pure protein sample required to start crystallization experiments. Ular purification from the tag protein was always performed in the presence of a 35 mer dsdna (o2+15) encompassing the sequence of an operator and half or with ular bound to the l-ascorbate-6-phophate molecule (the ula inducer, determined in this work). The crystallization of those complexes was tested and crystals were observed for both complexes. Only the complex ular-o2+15 produced positive diffraction results. However, the obtained diffraction data were not sufficient to accomplish the structure determination. directed mutagenesis of several residues of ular was undergone after the identification of well conserved amino acids across the deor family members. These amino acids are located in the n-terminal and c-terminal domains and they should play an important role in dna and/or inducer binding. Site-directed mutagenesis experiments allowed the identification of arg6, arg37, arg38, asp39, and arg53 as residues involved in dna recognition, and asp206 and lys209 in l-ascorbate-6-p recognition. the biophysical experiments like isothermal titration calorimetry and analytical ultracentrifugation with ular bound to its specifics ligands (cognate dna and inducer molecule) aims the identification of possible quaternary transitions that ular could adopt when bound to dna or to the effector molecule. These results allowed us to propose the first regulation model for a member of the deor family. Thus, analytical ultracentrifugation experiments of ular bound to dna show a tetramer conformation, whereas when bound to l-ascorbate-6-p this protein shows a dimeric organization. Isothermal titration calorimetric results also confirmed the dimeric organization of ular in the presence of its inducer, establishing a stoichiometry of 2:1 (two ular molecules to one of l-ascorbate-6-p). Concluding, ular binds dna in a tetrameric conformation establishing the ula repressed state whereas binding of l-ascorbate-6-p induces the quaternary rearrangement of ular to a dimer, which results in the breakdown of ular-dna complex and allows transcription of ulag and ulaabcdef structural genes. we determined the crystal structure of ulag, the first enzyme in the l-ascorbate-6-p dissimilation pathway at 2.5 í¥ by single anomalous diffraction (sad) method. Structure of ulag has the typical zinc-dependent hydrolase folding that belongs to metallo íY-lactamase superfamily. Structural homology comparisons show a relation between the fold of the ulag monomer and that of enzymes involved in rna metabolism, including trna maturation. This relationship indicates that ulag shares a common ancestor with members of íY-lactamase superfamily with rnase activity. However, ulag presents added structural features that allow a higher oligomerization level in the quaternary structure while assuring the integrity of the active site which has evolved towards a specialised function from the ancestral íY-lactamase.
Datos académicos de la tesis doctoral «Structural and biochemical characterization of ular and ulag proteins involved in l-ascorbate metabolism in escherichia coli«
- Título de la tesis: Structural and biochemical characterization of ular and ulag proteins involved in l-ascorbate metabolism in escherichia coli
- Autor: Fernando Oscar Garcés Ferreira
- Universidad: Barcelona
- Fecha de lectura de la tesis: 04/12/2008
Dirección y tribunal
- Director de la tesis
- Laura Baldoma Llavines
- Tribunal
- Presidente del tribunal: miquel Coll capella
- Francisco José Fernández pérez (vocal)
- joaquim Ros salvador (vocal)
- Santiago Imperial ródenas (vocal)