Characterization of aquacobalamin reductase (NADPH) from Euglena gracilis.
Aquacobalamin reductase (NADPH), which catalyzes the reduction of aquacobalamin to cob(II)alamin in the synthesis of cobalamin coenzymes, has already been purified from mitochondria of Euglena gracilis and partly characterized. Here, the enzyme was further characterized to clarify its enzymatic properties. The enzyme reduced 2 mol of aquacobalamin per mole of NADPH and had NADPH diaphorase-like activity. The 16 amino acid residues at the NH2-terminal of the enzyme were identical with those of the NADPH diaphorase domain of pyruvate: NADP+ oxidoreductase, which is involved in Euglena wax ester fermentation. Peptide mapping of the aquacobalamin reductase showed that elution during C-18 reversed-phase high-performance liquid chromatography was identical to that of the NADPH diaphorase domain. Immunoblotting indicated that the Euglena aquacobalamin reductase had a higher molecular weight (166,000) in the intact mitochondria than the purified enzyme (65,000), and that the molecular weights of the native and purified enzyme were identical with those of the subunit and the NADPH diaphorase domain, respectively. These results showed that the aquacobalamin reductase isolated earlier was the NADPH diaphorase domain, cleaved by trypsin during preparation of the mitochondrial homogenate from the native enzyme. Purified pyruvate:NADP+ oxidoreductase also had the activity of aquacobalamin reductase, which suggests that the enzyme in Euglena mitochondria has more than one function in the synthesis of cobalamin co-enzymes.
Watanabe
F
F
Laboratory of Nutrition and Food Science, Hagoromo-gakuen College, Osaka, Japan.
Yamaji
R
R
Isegawa
Y
Y
Yamamoto
T
T
Tamura
Y
Y
Nakano
Y
Y
D000818:Animals; D005056:Euglena gracilis / Q000201:enzymology*; D005779:Immunodiffusion; D008928:Mitochondria / Q000201:enzymology; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D013379:Substrate Specificity; D014805:Vitamin B 12 / Q000031:analogs & derivatives / Q000378:metabolism
C043960:aquacobalamin; C068157:aquacobalamin reductase; D009247:NADH, NADPH Oxidoreductases; D014805:Vitamin B 12