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 C 4 synthase (LTC 4 ), 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 ⁄ LTC 4 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 lmolAEmin )1 AEmg)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 lmolAEmin )1 AEmg )1 . Yeast microsomes expressing the Arabidopsis enzyme Abbreviations BSA, bovine serum albumin;

Modified forms of the D1 protein with deletions in lumen-exposed regions, were constructed in the cyanobacterium Synechocystis 6803 using site-directed mutagenesis. Integration and stability of the mutated D1 proteins in the thylakoid membrane were studied by immunoblot and pulse-chase analyses. It was found that in Δ(N325-E333), the D1 protein with a deletion in the C-terminal tail, could insert in the thylakoids to normal amounts but its stability in the membrane was dramatically reduced. Insertion of D1 in Δ(V58-D61) or Δ(D103-G109);G110R, with deletions in the A-B loop, was severely obstructed, For Δ(P350-T354), with a deletion in the processed region of the C-terminus of D1, no phenotypic effects were observed. The effects of failed D1 insertion or accumulation on Photosystem II assembly was monitored by immunoblot analysis. The conclusions from these experiments are that the extrinsic 33 kDa protein, CP43, and the β subunit of cytochrome b559 accumulate in the thylakoid membrane independently of the D1 protein, and that accumulation of the D2 protein and CP47 requires insertion but not necessarily accumulation of the D1 protein.

No abstract

Mutants of the cyanobacterium Synechocystis sp. PCC 6803 with N-terminal changes in the photosystem (PSII) II D1 protein were analysed by flash-induced oxygen evolution, chlorophyll a fluorescence decay kinetics and 77 K fluorescence emission spectra. The data presented here show that mutations of the Thr-2, Thr-3 and Thr-4 in D1 do not influence the oxygen evolution. A perturbation on the acceptor side was observed and the importance of the N-terminal threonines for an efficient energy transfer between the phycobilisome and PSII and for stability of the PSII complex was demonstrated.z 1998 Federation of European Biochemical Societies.

One prominent difference between the photosystem II (PSII) reaction center protein D1P P in Synechocystis 6803 and normal D1 is the replacement of Phe-186 in D1 with leucine in D1P P. Mutants of Synechocystis 6803 producing only D1P P, or containing engineered D1 proteins with Phe-186 substitutions, were analyzed by 77 K fluorescence emission spectra, chlorophyll a fluorescence induction yield and decay kinetics, and flashinduced oxygen evolution. Compared to D1-containing PSII centers, D1P P centers exhibited a 50% reduction in variable chlorophyll a fluorescence yield, while the flash-induced O 2 evolution pattern was unaffected. In the F186 mutants, both the P680 + /Q Â recombination and O 2 oscillation pattern were noticeably perturbed. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.