Abstract
Homogeneous ferredoxin (flavodoxin):NADP(+) reductase and flavodoxin A proteins served as electron donors for the reduction of co(III)rrinoids to co(I)rrinoids in vitro. The resulting co(I)rrinoids served as substrates for the ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica serovar Typhimurium LT2 and were converted to their respective adenosylated derivatives. The reaction products were isolated by reverse phase high performance liquid chromatography, and their identities were confirmed by UV-visible spectroscopy, mass spectrometry, and in vivo biological activity assays. Adenosylcobalamin generated by this system supported the activity of 1,2-propanediol dehydratase as effectively as authentic adenosylcobalamin. This is the first report of a protein system that can be coupled to the adenosyltransferase CobA enzyme for the conversion of co(III)rrinoids to their adenosylated derivatives.
Highlights
Ferase (EC 2.5.1.17) (CobA in Salmonella enterica)
The data reported show that the reducing system comprised of the Fpr and FldA proteins is sufficient for the generation of the co(I)rrinoid substrate of the ATP:co(I)rrinoid adenosyltransferase CobA enzyme of S. enterica
It is reasonable to think that reduced FldA might represent a natural reducing system for corrinoid adenosylation in enterobacteria (e.g. E. coli and S. enterica)
Summary
Step 1: Mass Culturing of the Overexpressing Strain and Generation of Cell-free Extracts—Fpr protein was overproduced from strain JE4937 (E. coli C-1a/pEE1010, a gift from Elisabeth Haggård-Ljungquist, Stockholm University, Stockholm, Sweden) grown overnight at 37 °C on Luria Bertani broth containing 100 g/ml of ampicillin and 30 mM glucose. The cells were harvested by centrifugation at 10,000 ϫ g for 10.
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