An in vitro reducing system for the enzymic conversion of cobalamin to adenosylcobalamin.
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.
Fonseca
M V
MV
Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Escalante-Semerena
J C
JC
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003038:Cobamides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D005418:Flavodoxin / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017386:Sequence Homology, Amino Acid; D014805:Vitamin B 12 / Q000378:metabolism*
D001426:Bacterial Proteins; D003038:Cobamides; D005418:Flavodoxin; D005287:Ferredoxin-NADP Reductase; D019883:Alkyl and Aryl Transferases; C082269:ATP-corrinoid adenosyltransferase; C000913:cobamamide; D014805:Vitamin B 12