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Bioinformatic and enzymatic characterization of the MAPEG superfamily
Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology.
Department of Medicine, Division of Rheumatology Unit, Karolinska Institutet, Stockholm.
Department of Plant Biology & Forestry Genetics, Swedish Agricultural University, Uppsala.
Department of Plant Biology & Forestry Genetics, Swedish Agricultural University, Uppsala.
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2005 (English)In: The FEBS Journal, ISSN 1742-464X, Vol. 272, no 7, 1688-1703 p.Article in journal (Refereed) Published
Abstract [en]

The membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily includes structurally related membrane proteins with diverse functions of widespread origin. A total of 136 proteins belonging to the MAPEG superfamily were found in database and genome screenings. The members were found in prokaryotes and eukaryotes, but not in any archaeal organism. Multiple sequence alignments and calculations of evolutionary trees revealed a clear subdivision of the eukaryotic MAPEG members, corresponding to the six families of microsomal glutathione transferases (MGST) 1, 2 and 3, leukotriene C4 synthase (LTC4), 5-lipoxygenase activating protein (FLAP), and prostaglandin E synthase. Prokaryotes contain at least two distinct potential ancestral subfamilies, of which one is unique, whereas the other most closely resembles enzymes that belong to the MGST2/FLAP/LTC4 synthase families. The insect members are most similar to MGST1/prostaglandin E synthase. With the new data available, we observe that fish enzymes are present in all six families, showing an early origin for MAPEG family differentiation. Thus, the evolutionary origins and relationships of the MAPEG superfamily can be defined, including distinct sequence patterns characteristic for each of the subfamilies. We have further investigated and functionally characterized representative gene products from Escherichia coli, Synechocystis sp., Arabidopsis thaliana and Drosophila melanogaster, and the fish liver enzyme, purified from pike (Esox lucius). Protein overexpression and enzyme activity analysis demonstrated that all proteins catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene with reduced glutathione. The E. coli protein displayed glutathione transferase activity of 0.11 µmol·min−1·mg−1 in the membrane fraction from bacteria overexpressing the protein. Partial purification of the Synechocystis sp. protein yielded an enzyme of the expected molecular mass and an N-terminal amino acid sequence that was at least 50% pure, with a specific activity towards 1-chloro-2,4-dinitrobenzene of 11 µmol·min−1·mg−1. Yeast microsomes expressing the Arabidopsis enzyme showed an activity of 0.02 µmol·min−1·mg−1, whereas the Drosophila enzyme expressed in E. coli was highly active at 3.6 µmol·min−1·mg−1. The purified pike enzyme is the most active MGST described so far with a specific activity of 285 µmol·min−1·mg−1. Drosophila and pike enzymes also displayed glutathione peroxidase activity towards cumene hydroperoxide (0.4 and 2.2 µmol·min−1·mg−1, respectively). Glutathione transferase activity can thus be regarded as a common denominator for a majority of MAPEG members throughout the kingdoms of life whereas glutathione peroxidase activity occurs in representatives from the MGST1, 2 and 3 and PGES subfamilies.

Place, publisher, year, edition, pages
2005. Vol. 272, no 7, 1688-1703 p.
Keyword [en]
MAPEG, microsomal glutathione transferase, prostaglandin, leukotriene
National Category
Natural Sciences
URN: urn:nbn:se:liu:diva-12886DOI: 10.1111/j.1742-4658.2005.04596.xOAI: diva2:17295
Available from: 2008-01-28 Created: 2008-01-28 Last updated: 2009-11-07Bibliographically approved
In thesis
1. Characterization of protein families, sequence patterns, and functional annotations in large data sets
Open this publication in new window or tab >>Characterization of protein families, sequence patterns, and functional annotations in large data sets
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Bioinformatics involves storing, analyzing and making predictions on massive amounts of protein and nucleotide sequence data. The thesis consists of six papers and is focused on proteins. It describes the utilization of bioinformatics techniques to characterize protein families and to detect patterns in gene expression and in polypeptide occurrences. Two protein families were bioinformatically characterized - the membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) and the Tripartite motif (TRIM) protein families.

In the study of the MAPEG super-family, application of different bioinformatic methods made it possible to characterize many new members leading to a doubling of the family size. Furthermore, the MAPEG members were subdivided into families. Remarkably, in six families with previously predominantly mammalian members, fish representatives were also now detected, which dated the origin of these families back to the Cambrium ”species explosion”, thus earlier than previously anticipated. Sequence comparisons made it possible to define diagnostic sequence patterns that can be used in genome annotations. Upon publication of several MAPEG structures, these patterns were confirmed to be part of the active sites.

In the TRIM study, the bioinformatic analyses made it possible to subdivide the proteins into three subtypes and to characterize a large number of members. In addition, the analyses showed crucial structural dependencies between the RING and the B-box domains of the TRIM member

Ro52. The linker region between the two domains, denoted RBL, is known

to be disease associated. Now, an amphipathic helix was found to be a

characteristic feature of the RBL region, which also was used to divide the family into three subtypes.

The ontology annotation treebrowser (OAT) tool was developed to detect functional similarities or common concepts in long lists of proteins or genes, typically generated from proteomics or microarray experiments. OAT was the first annotation browser to include both Gene Ontology (GO) and Medical Subject Headings (MeSH) into the same framework. The complementarity of these two ontologies was demonstrated. OAT was used in the TRIM study to detect differences in functional annotations between the subtypes.

In the oligopeptide study, we investigated pentapeptide patterns that were over- or under-represented in the current de facto standard database of protein knowledge and a set of completed genomes, compared to what could be expected from amino acid compositions. We found three predominant categories of patterns: (i) patterns originating from frequently occurring families, e.g. respiratory chain-associated proteins and translation machinery proteins; (ii) proteins with structurally and/or functionally favored patterns; (iii) multicopy species-specific retrotransposons, only found in the genome set. Such patterns may influence amino acid residue based prediction algorithms. These findings in the oligopeptide study were utilized for development of a new method that detects translated introns in unverified protein predictions, which are available in great numbers due to the many completed and ongoing genome projects.

A new comprehensive database of protein sequences from completed genomes was developed, denoted genomeLKPG. This database was of central importance in the MAPEG, TRIM and oligopeptide studies. The new sequence database has also been proven useful in several other studies.

Place, publisher, year, edition, pages
Institutionen för fysik, kemi och biologi, 2008. 85 p.
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1159
Bioinformatics, sequence analysis, patterns, protein families
National Category
Bioinformatics (Computational Biology)
urn:nbn:se:liu:diva-10565 (URN)978-91-85523-01-6 (ISBN)
Public defence
2008-02-15, Planck, Fysikhuset, Linköpings Universitet, Linköping, 10:15 (English)
Available from: 2008-01-28 Created: 2008-01-28 Last updated: 2010-01-13Bibliographically approved

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