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- Bresell, Anders, et al.
(författare)
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Bioinformatic and enzymatic characterization of the MAPEG superfamily
- 2005
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 272:7, s. 1688-1703
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Weinander, Rolf
(författare)
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Microsomal glutathione transferase : catalysis, in vitro mutagenesis and heterologous expression
- 1996
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Microsomal glutathione transferase is a membrane-bound member of the glutathione transferases, a family of multifunctional enzymes involved in the cellular detoxification of xenobiotics and reactive endogenous compounds formed during oxidative stress. Rat liver microsomal glutathione transferase is a trimeric protein with a molecular mass of 17.3 kDa per subunit which displays unique features regarding DNA and amino acid sequence, molecular weight, enzymatic properties and ability of activation by various agents as compared to its cytosolic counterparts. At the outset of this work we were interested in obtaining: specific substrates and expression systems for the enzyme. These experimental tools combined with in vitro mutagenesis were used to obtain information on the functional role of individual amino acids as well as membrane topology. N-acetyl-L-cysteine was found to serve as a substrate for the microsomal glutathione transferase (with l-chloro-2,4-dinitrobenzene (CDNB) as second substrate). In examining the activity of liver subcellular fractions, no activity with N-acetyl-L-cysteine could be detected in cytosols devoid of endogenous glutathione. Thus, N-acetyl-L-cysteine is a specific substrate for microsomal glutathione transferase. The pH dependence of kCat/Km(CDNB) for the microsomal glutathione transferase with different thiol substrates indicates that the enzyme has the ability to lower the pKa of bound glutathione by 3 orders of magnitude. The microsomal glutathione transferase stabilizes Meisenheimer complex formation between 1,3,5-trinitrobenzene and various glutathione analogues, including some non-substrate thiols, thus offering new possibilities for examining ligand interactions of glutathione transferases. Rat liver microsomal glutathione transferase was successfully expressed both in mammalian COS-cells and in E. Coli BL 21 (DE3). Significant amounts of enzymatically active protein was expressed in the inner membrane of this E. Coli strain. Recombinant rat microsomal glutathione transferase was purified from bacterial membranes and was found to be indistinguishable from the liver enzyme with regard to enzymatic activity, molecular mass, immunoreactivity and N-terminal amino acid sequence. Chemical modification of rat liver microsomal glutathione transferase indicated that arginine 107 and Iysine 67 are essential for enzyme activity and may thus reside in the active site. A set of mutant forms of the rat enzyme were constructed by site-directed mutagenesis and heterologously expressed in E. coli BL21(DE3). Arginine 107 was exchanged for alanine and Iysine residues. The alanine mutant (R107A) displayed a decreased thermostability and an important structural role is suggested for this residue. Neither mutation of Iysine 67 to alanine and arginine nor replacement of the three histidines by glutamines yielded any drastic changes of activity in contrast to the chemical modification experiments. All tyrosine to phenylalanine substitutions resulted in mutants with activities similar to the wild type. Thus, the microsomal glutathione transferase must perform an alternate stabilization of the thiolate anion of glutathione than through interaction with a phenolic hydroxyl group of a tyrosine. Substitution of cysteine 49 with alanine resulted in a semi-activated mutant enzyme which was not affected by N-ethylmaleimide. Cysteine 49 is therefore unambiguously demonstrated as the site of modification that results in activation of microsomal glutathione transferase. The membrane topology of rat liver microsomal glutathione transferase was investigated by in situ proteolysis of intact and permeabilized rat liver microsomes. Lysine 4 is accessible at the luminal surface of the endoplasmic reticulum, whereas Iysine 41 faces the cytosol. These positions are separated by a hydrophobic stretch of 25 amino acids which comprises a likely membrane-spanning region. The cytosolic location of the active site was demonstrated using radiolabeled glutathione. Additional membrane anchoring(s) are indicated since the C-terminal part of the trypsin-cleaved protein was not separated from the membrane fraction by intensive salt washing or phase separation in Triton X-l 14. Cleavage of the purified protein at Iysine 41 and subsequent separation of the fragments yielded a catalytically competent C-terminal polypeptide. The topology of recombinant rat microsomal glutathione transferase expressed in E. coli was investigated by comparing the proteolytic cleavage products from intact and permeabilized spheroplasts. Tryptic cleavage at Iysine 4 in intact spheroplasts shows that this residue is directed towards the periplasmic side, whereas Iysine 41 faces the inside of the E. Coli inner membrane. Intact spheroplasts treated with pronase yielded a cleavage pattern consistent with two additional C-terminal sites exposed to the periplasmic side indicating a polytopic membrane association of microsomal glutathione transferase.
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