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The advantages of being small: Glycosyltransferases in many dimensions and glycolipid synthesis in Mycoplasma Pneumoniae
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The synthesis and breakdown of sugars is one of the most important functions in Nature. Consequently, sugar structures are used both as energy storage and as building blocks to stabilise and protect the cell. The formation of these structures is performed by glycosyltransferases (GT), an enzyme group structurally conserved within all kingdoms. Until now, only two different folds have been discovered by crystallisation of GTs, i.e. GT-A and GT-B. A third fold family has however been proposed by fold predictions. In this thesis, a multivariate data analysis was successfully used in classifying and predicting both fold and reaction mechanism (inverting or retaining) of GTs. This method was also used to obtain information about the separating parameters for the reaction mechanism classification. This information could be traced back to the amino acid sequence. The method could as well be used to analyse and identify the properties of membrane binding regions of GTs, and subsequently distinguish soluble from membrane-associated enzymes. Most functionally characterised enzymes only use one substrate, synthesising one product. Mycoplasma pneumoniae, a common human pathogen with a small genome has only three proposed GTs. The bacterium was, however expected to have a greater number of GTs, due to its ability to make both glycolipids and capsule. Here we have determined the function of one of these enzymes, MPN483 and discovered its ability to both use different acceptors, and make elongated glycolipids with up to three galactose residues, with both DAG and ceramide as the base. Many of the synthesised glycolipids were also found to be immunogenic, hence showing their biological importance. The properties of lipids are known to be important for the function of a biological membrane. We have here shown that not only the charge but also the shape of the lipids are important for several protein mediated membrane processes in Echerichia coli, such as the function of the LacY.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik , 2007. , 56 p.
Keyword [en]
Glycosyltransferase, lipid, membrane, glycolipid, ACC, PLS-DA
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-7056ISBN: 978-91-7155-503-8 (print)OAI: oai:DiVA.org:su-7056DiVA: diva2:197569
Public defence
2007-10-05, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2007-09-13 Created: 2007-09-04Bibliographically approved
List of papers
1. Recognition of fold and sugar linkage for glycosyltransferases by multivariate sequence analysis
Open this publication in new window or tab >>Recognition of fold and sugar linkage for glycosyltransferases by multivariate sequence analysis
2004 In: J Biol Chemistry, ISSN 0021-9258, Vol. 279, no 37, 38683-92 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24459 (URN)
Note
Part of urn:nbn:se:su:diva-7056Available from: 2007-09-13 Created: 2007-09-04Bibliographically approved
2. High cationic charge and bilayer interface-binding helices in a regulatory lipid glycosyltransferase
Open this publication in new window or tab >>High cationic charge and bilayer interface-binding helices in a regulatory lipid glycosyltransferase
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2007 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 19, 5664-5677 p.Article in journal (Refereed) Published
Abstract [en]

In the prokaryote Acholeplasma laidlawii, membrane bilayer properties are sensed and regulated by two interface glycosyltransferases (GTs), synthesizing major nonbilayer- (alMGS GT) and bilayer-prone glucolipids. These enzymes are of similar structure, as many soluble GTs, but are sensitive to lipid charge and curvature stress properties. Multivariate and bioinformatic sequence analyses show that such interface enzymes, in relation to soluble ones of similar fold, are characterized by high cationic charge, certain distances between small and cationic amino acids, and by amphipathic helices. Varying surface contents of Lys/Arg pairs and Trp indicate different membrane-binding subclasses. A predicted potential (cationic) binding helix from alMGS was structurally verified by solution NMR and CD. The helix conformation was induced by a zwitterionic as well as anionic lipid environment, and the peptide was confined to the bilayer interface. Bilayer affinity of the peptide, analyzed by surface plasmon resonance, was higher than that for soluble membrane-seeking proteins/peptides and rose with anionic lipid content. Interface intercalation was supported by phase equilibria in membrane lipid mixtures, analyzed by 31P NMR and DSC. An analogous, potentially binding helix has a similar location in the structurally determined Escherichia coli cell wall precursor GT MurG. These two helices have little sequence conservation in alMGS and MurG homologues but maintain their amphipathic character. The evolutionary modification of the alMGS binding helix and its location close to the acceptor substrate site imply a functional importance in enzyme catalysis, potentially providing a mechanism by which glycolipid synthesis will be sensitive to membrane surface charge and intrinsic curvature strain.

Identifiers
urn:nbn:se:su:diva-24460 (URN)10.1021/bi700042x (DOI)000246283600005 ()
Note
Part of urn:nbn:se:su:diva-7056Available from: 2007-09-13 Created: 2007-09-04 Last updated: 2017-12-13Bibliographically approved
3. A processive lipid glycosyltransferase in the small human pathogen Mycoplasma pneumoniae: Involvement in host immune response
Open this publication in new window or tab >>A processive lipid glycosyltransferase in the small human pathogen Mycoplasma pneumoniae: Involvement in host immune response
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2007 In: Molecular Microbiology, ISSN 0950-382X, Vol. 65, no 6, 1444-57 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24461 (URN)000249425600008 ()
Note
Part of urn:nbn:se:su:diva-7056Available from: 2007-09-13 Created: 2007-09-04Bibliographically approved
4. Curvature-engineered Escherichia coli bilayers reveal critical lipid head-group size for membrane protein function in vivo
Open this publication in new window or tab >>Curvature-engineered Escherichia coli bilayers reveal critical lipid head-group size for membrane protein function in vivo
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Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-24462 (URN)
Note
Part of urn:nbn:se:su:diva-7056Available from: 2007-09-13 Created: 2007-09-04 Last updated: 2010-01-13Bibliographically approved

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