B12 METABOLISM IN HUMANS By NICOLE AURORA LEAL A ...

tetanomorphum, P. shermanii, Euglena gracilis, human leukocytes, human skin
fibroblasts, rat liver, and rabbit spleen (Weissbach et al. 1961, Brady et al. 1962,
Watanabe et al. 1987, Pezacka et al. 1990, Pezacka 1993). The enzymes involved and
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their encoding genes have not been identified. However, it was determined that for the
enzymatic conversion of CNCbl to AdoCbl, cell extracts required supplementation with
adenosine triphosphate (ATP), reduced flavin, and reduced glutathione (Weissbach et al.
1962). Studies in mammalian cells suggest that GSCbl is a product of the β-ligand
transferase reaction (Pezacka et al. 1990). In bacteria, this GSCbl intermediate has not
been found.
Cob(III)alamin reductase
The second enzymatic step in cobalamin metabolism is cob(III)alamin reduction.
Reductase activity has been detected in crude cell extracts of C. tetanomorphum,
P. shermanii, E. gracilis, human, and rat (Weissbach et al. 1961, Brady et al. 1962,
Watanabe et al. 1987, Pezacka et al. 1990, Pezacka 1993). Extracts of C. tetanomorphum
required NADH and either flavin mononucleotide (FMN) or flavin adenine dinucleotide
(FAD) to mediate reductase activity (Walker et al. 1969). The flavin oxidoreductase
(Fre) of S. enterica was purified and shown to mediate the nonenzymatic reduction of
cobalamin in vitro (Fonseca and Escalante-Semerena 2000). Fre reduces flavin
nucleotides, which in turn chemically reduces cob(III)alamin. In E. gracilis,
cob(III)alamin reductase activity was purified from the mitochondrial fraction, and was
ultimately identified as a flavoprotein with an absolute requirement for NADPH but not
FAD or FMN (Watanabe et al. 1987). Further analysis of this protein revealed that it
contained one molecule of either FAD or FMN that remained bound during the