Tesis durant el mes de setembre a la URL

Universitat Ramon LlullJavier Antonio Linares Pastén, Eduardo Andrés Marínez i Marc Saura Valls han assolit el títol de Doctor a la URL durante el mes de setembre:


Javier Antonino Linares Pastén (Institut Químic de Sarrià) ha defensat la tesi que porta per títol Análisis mutacional del extremo c-terminal de la α-1,3-galactosiltransferasa bovina: estabilidad y catálisis, amb qualificació Excel.lent Cum Laude, i dirigida pel Dr. Antoni Planas Sauter:

Mammalian α-1,3-Galactosyltransferase (α3GT) is involved in the biosynthesis of the oligosaccharide antigen responsible for hiperacute (vascular) rejection (HAR) in xenotransplantation of animal organs to humans [1]. α3GT is also used as a model enzyme for studies of glycosyltransferase structure and mechanisms, as well as biocatalyst in enzymatic oligosacaride synthesis. The enzyme catalyzes the transfer of galactose (Gal) from UDP-Gal a la N-acetyllactosamyne (NAcLac), or to lactose.

Currently, there are two types of α3GT 3-dimensional structure. The previously reported form I [2] is similar to the form II [3,4], except at the C-terminal residues 358-368. The C-terminal region is highly disordered in form I but well defined in form II, suggesting that it functions as a lid that closes the active site. The conformational change at the C-terminus and the sequence conservation among other α3GT suggest that this region could have key role in the catalytic action [4]. 

In the present Doctoral Thesis, the study structure/function of the end C-terminal was approached by a kinetic analysis and by stability analysis of alanine mutants for each one of the amino acids at positions 358-368.  This way, the comparison of catalytic efficiency and enzymatic stability provides a method that allows identification of the amino acids that have an important role in the substrate binding or in structural packaging.

For mutants K359A, Y361A, V363A and R365A no activity was detected, therefore these positions are fundamental for α3GT activity. K359, Y361 and R365 interact with the UDP-Gal phosphates, also, K359A interacts with the 3-hydroxyl of the lactose according to molecular modeling, thereby it has been proposed to have the role as the general acid/base. On the other hand, the mutants N367A and V368A  turned out to be 2 or 3 times more active than wt, respectively. 

The studies of stability reveal that amino acids mutants (wt including) which don’t directly interact with the substrates do not present higher stability than the amino acid mutants which directly interact with the substrate UDP or UDP-Gal. On the other hand, the presence of UDP stabilizes de wt enzyme and the mutants of the amino acids that don’t directly interact with the substrates, while this UDP stabilizing effect diminishes in mutants of amino acids directly implied in the interactions of UDP or UDP-Gal.

Marc Saura Valls (Institut Químic de Sarrià) ha defensat la tesi que porta per títol Kinetic Studies of a xyloglucan endotransglycosylase, a key enzyme in plant cell morphogenesis, amb qualificació Excel.lent Cum Laude, i dirgida pel Dr. Antoni Planas Sauter:

The present work is part of an European project named E.D.E.N. (Enzyme Discovery in hybrid aspen for fibre ENgineering, QLK5-CT-2001-00443). The general objective of the project is to identify novel plant enzymes for deeper understanding of the process of fiber formation and modification for future improvement of the quality parameters of wood fibers. The present project pretends to increase the knowledge about xyloglucan endotransglycosylases (XET), which are thought to be key enzymes in the construction and controlled modification of the xyloglucan¬cellulose network. It is pretended to study the mechanism of action and the substrate specificity of a XET from Populus tremula x tremuloides, concretely XET16A (Ptt-XET16A).

A new enzymatic assay based on capillary electrophoresis is designed and validated. This assay allows the kinetic study of XETs using as substrates, low molecular mass xyloglucan oligosaccharides with defined structures. These substrates have been synthesized in the present work and also in collaboration with Dr. Driguez team from CERMAV-CNRS.

It is concluded that the maximum of activity of Ptt-XET16A is between pH 5 and 5.5 and 30 and 40 ºC. It is demonstrated that Ptt-XET16A follows a bi-bi ping-pong kinetic mechanism, in which the acceptor acts as competitive inhibitor of the donor binding to the free enzyme and depending on the donor used, this one can act also as competitive inhibitor of the acceptor binding to the acceptor subsites of the glycosyl-enzyme intermediate giving rise to side reaction such as donor polymerization and product elongation only in case that the donor shows a glucosyl residue in the non reducing end.

A library of xylogluco-oligosaccharides, synthesized in CERMAV-CNRS by Dr. Driguez team, is evaluated as Ptt-XET16A donors. With this studies we are able to deeper understand the activity of XETs, their substrate specificity and a subsite maping of the binding cleft is done, obtaining the contribution of different subsites of Ptt-XET16A to the stabilization of the transition state of the transglycosylation reaction catalyzed by the studied enzyme.

Finally, a bifluorogenic substrate derived from the tetradecasacharide used as standard substrate in this project has been designed to measure hydrolase and transferase activities of XET enzymes by fluorescense resonance energy transfer (FRET). The bifluorogenic substrate was obtained, however, it could not be demonstrated if it is an adequate substrate to measure hydrolase and transferase activities because the fluorescent properties of the label were lost during substrate synthesi.

Eduardo Andrés Martínez  (Institut Químic de Sarrià) ha defensat la tesi que porta per títol Glicosiltransferasas en la biosíntesis de glicolípidos de membrana de mycoplasma genitalium, amb qualificació Excel.lent Cum Laude, i dirgida pel Dr. Antoni Planas Sauter:

Mycoplasma are gram positive bacteria whose parasite life allowed the lost of genomic material in a deleterious evolution missing most of its catabolic pathways and biosynthetic capabilities; including the cell wall synthesis and having thereby the membrane as the solely barrier.

This metabolic reduction and decrease of genome size stated Mycoplasma genitalium (extreme example of this simplification of cellular functions) as a model organism in the development of  genomics and proteomics. Being also the second genome completely sequenced, it has been used as tarting point in the research for the minimal gene set needed for life, under the hypothesis that “The machinery that perpetuates live is finite, relatively simple and can be subject of exploration and full understanding”.

Besides this theoretical interest, in its pathogenic life this organisms are involved in different pathologies (pneumonia, arthritis, etc.). Always as opportunistic pathogens, acting on systems hose defences have been reduced; and being also the most common contaminant of clinical iopsies. The main problem arises from its immunity of these organisms against certain antibiotics β-lactamic, rifampicin). This lack of specific antibiotics, merged with a difficult immune response once mycoplasma has infected and located inside host cells, makes mycoplasma infections hard to treat and remove.

The present work starts the study of membrane glycolipids of M.genitalium (and the lycosiltransferases involved in their synthesis) as possible therapeutic targets for the specific reatment of these organisms. Lacking a cell wall, the mycoplasma base their structural integrity in close regulation of membrane glycolipids for keeping the fluidity that allows its functionality, but aintaining it rigid enough to accomplish its structural function.

Checking first the existence of a metabolic pathway for the synthesis of glycolipids, carried out by glycosyltransferases, through the identification of both lipids and activities in M.genitalium. Proceeding afterwards to search and clone on the M.genitalium genome those ORFs (mg025, mg060 y mg335.2) that could potentially be responsible of these activities until its finalo ntroduction into the constructions for the expression of each of these ORFs in E.coli.

By the expression of the cloned genes, mg335.2 was identified as the only responsible of both glycolipids biosynthesis; witch appeared in the corresponding E.coli strain. Finally the purification of mg335.2 protein could be achieved through  chromatographic techniques.

Glycolipids were purified from recombinant E.coli and also enzymatically synthesized with the purified protein. Both were analyzed by NMR techniques which determined the structure of the polar head as 1 – (glucopiranosil – β (1→6) – glucopiranosil) diacil – sn – glycerol, different of the other glycolipid structure known in the mycoplasma A.laidlawii of α (1→2) connectivity, but more representative attending to bioinformatic analysis of the sequenced genomes.


+ info: TDX Javier Antonino Linares Pastén, TDX Marc Saura Valls