Tài liệu Báo cáo khoa học: Bioinformatic and enzymatic characterization of the MAPEG superfamily doc

Bioinformatic and enzymatic characterization

of the MAPEG superfamily

Anders Bresell1,*, Rolf Weinander2,*, Gerd Lundqvist3

, Haider Raza3

, Miyuki Shimoji3

,

Tie-Hua Sun3

, Lennart Balk5

, Ronney Wiklund6

, Jan Eriksson6

, Christer Jansson6

, Bengt Persson1,4,

Per-Johan Jakobsson2 and Ralf Morgenstern3

1 IFM Bioinformatics, Linko¨ping University, Sweden

2 Department of Medicine, Division of Rheumatology Unit, Karolinska Institutet, Stockholm, Sweden

3 Institute of Environmental Medicine Karolinska Institutet, Stockholm, Sweden

4 Centre for Genomics and Bioinformatics, Karolinska Institutet, Stockholm, Sweden

5 Stockholm Marine Research Centre, University of Stockholm, Sweden

6 Department of Plant Biology & Forestry Genetics, Swedish Agricultural University, Uppsala, Sweden

Keywords

MAPEG; microsomal glutathione

transferase; prostaglandin; leukotriene

Correspondence

R. Morgenstern, Institute of Environmental

Medicine, Karolinska Institutet, S-171 77

Stockholm, Sweden

Fax: +46 8 343849

Tel: +46 8 5248 7574

E-mail: [email protected]

*Both authors contributed equally to this

work

(Received 15 November 2004, revised 27

January 2005, accepted 3 February 2005)

doi:10.1111/j.1742-4658.2005.04596.x

The membrane associated proteins in eicosanoid and glutathione metabo￾lism (MAPEG) superfamily includes structurally related membrane proteins

with diverse functions of widespread origin. A total of 136 proteins belong￾ing 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 calcula￾tions of evolutionary trees revealed a clear subdivision of the eukaryotic

MAPEG members, corresponding to the six families of microsomal gluta￾thione 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, show￾ing an early origin for MAPEG family differentiation. Thus, the evolution￾ary origins and relationships of the MAPEG superfamily can be defined,

including distinct sequence patterns characteristic for each of the sub￾families. We have further investigated and functionally characterized repre￾sentative 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 ana￾lysis 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 lmolÆmin)1

Æmg)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 lmolÆmin)1

Æmg)1

. Yeast microsomes expressing the Arabidopsis enzyme

Abbreviations

BSA, bovine serum albumin; CDNB, 1-chloro-2,4-dinitrobenzene; DEAE, diethylaminoethyl; FLAP, 5-lipoxygenase activating protein; LT,

leukotriene; MGST, microsomal glutathione transferase; PG, prostaglandin; PGES, prostaglandin E synthase; GST, glutathione S-transferase;

GPx, glutathione peroxidase; CuOOH, cumene hydroperoxide.

1688 FEBS Journal 272 (2005) 1688–1703 ª 2005 FEBS