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S-adenosyl-L-methionine:magnesium-protoporphyrin IX O-methyltransferase from Rhodobacter capsulatus: mechanistic insights and stimulation with phospholipids.
- Source :
-
The Biochemical journal [Biochem J] 2007 Sep 15; Vol. 406 (3), pp. 469-78. - Publication Year :
- 2007
-
Abstract
- The enzyme BchM (S-adenosyl-L-methionine:magnesium-protoporphyrin IX O-methyltransferase) from Rhodobacter capsulatus catalyses an intermediate reaction in the bacteriochlorophyll biosynthetic pathway. Overexpression of His(6)-tagged protein in Escherichia coli resulted in the majority of polypeptide existing as inclusion bodies. Purification from inclusion bodies was performed using metal-affinity chromatography after an elaborate wash step involving surfactant polysorbate-20. Initial enzymatic assays involved an in situ generation of S-adenosyl-L-methionine substrate using a crude preparation of S-adenosyl-L-methionine synthetase and this resulted in higher enzymatic activity compared with commercial S-adenosyl-L-methionine. A heat-stable stimulatory component present in the S-adenosyl-L-methionine synthetase was found to be a phospholipid, which increased enzymatic activity 3-4-fold. Purified phospholipids also stabilized enzymatic activity and caused a disaggregation of the protein to lower molecular mass forms, which ranged from monomeric to multimeric species as determined by size-exclusion chromatography. There was no stimulatory effect observed with magnesium-chelatase subunits on methyltransferase activity using His-BchM that had been stabilized with phospholipids. Substrate specificity of the enzyme was limited to 5-co-ordinate square-pyramidal metalloporphyrins, with magnesium-protoporphyrin IX being the superior substrate followed by zinc-protoporphyrin IX and magnesium-deuteroporphyrin. Kinetic analysis indicated a random sequential reaction mechanism. Three non-substrate metalloporphyrins acted as inhibitors with different modes of inhibition exhibited with manganese III-protoporphyrin IX (non-competitive or uncompetitive) compared with cobalt II-protoporphyrin IX (competitive).
Details
- Language :
- English
- ISSN :
- 1470-8728
- Volume :
- 406
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- The Biochemical journal
- Publication Type :
- Academic Journal
- Accession number :
- 17594291
- Full Text :
- https://doi.org/10.1042/BJ20070284