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, 284 (48), 33418-24

A Human Vitamin B12 Trafficking Protein Uses Glutathione Transferase Activity for Processing Alkylcobalamins

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A Human Vitamin B12 Trafficking Protein Uses Glutathione Transferase Activity for Processing Alkylcobalamins

Jihoe Kim et al. J Biol Chem.

Abstract

Pathways for tailoring and processing vitamins into active cofactor forms exist in mammals that are unable to synthesize these cofactors de novo. A prerequisite for intracellular tailoring of alkylcobalamins entering from the circulation is removal of the alkyl group to generate an intermediate that can subsequently be converted into the active cofactor forms. MMACHC, a cytosolic cobalamin trafficking chaperone, has been shown recently to catalyze a reductive decyanation reaction when it encounters cyanocobalamin. In this study, we demonstrate that this versatile protein catalyzes an entirely different chemical reaction with alkylcobalamins using the thiolate of glutathione for nucleophilic displacement to generate cob(I)alamin and the corresponding glutathione thioether. Biologically relevant thiols, e.g. cysteine and homocysteine, cannot substitute for glutathione. The catalytic turnover numbers for the dealkylation of methylcobalamin and 5'-deoxyadenosylcobalamin by MMACHC are 11.7 +/- 0.2 and 0.174 +/- 0.006 h(-1) at 20 degrees C, respectively. This glutathione transferase activity of MMACHC is reminiscent of the methyltransferase chemistry catalyzed by the vitamin B(12)-dependent methionine synthase and is impaired in the cblC group of inborn errors of cobalamin disorders.

Figures

SCHEME 1.
SCHEME 1.
Alternative reaction mechanisms for dealkylation of alkylcobalamin.
FIGURE 1.
FIGURE 1.
Reactions catalyzed by MMACHC. CNCbl in the presence of NADPH and an oxidoreductase is converted via a reductive decyanation reaction to cyanide and cob(II)alamin. Alternatively, alkylcobalamins undergo nucleophilic displacement to give cob(I)alamin, which is oxidized to cob(II)alamin and subsequently converted to the active cofactor forms for the vitamin B12-dependent enzymes methionine synthase (MS) and methylmalonyl-CoA mutase (MCM).
FIGURE 2.
FIGURE 2.
Dealkylation of MeCbl and AdoCbl bound to MMACHC under aerobic conditions. Shown are the spectra of MMACHC·MeCbl (60:40 μm; blue trace in a) and MMACHC·AdoCbl (60:40 μm; blue trace in b) in aerobic 100 mm HEPES (pH 8.0) containing 150 mm KCl and 10% glycerol. Addition of GSH (1 mm) resulted in the spectral changes that are consistent with formation of OH2Cbl. Insets show the absorbance increases at 355 nm (○) at 20 °C and the single exponential fits to the data (solid line).
FIGURE 3.
FIGURE 3.
HPLC analysis for alkyl transfer reaction products. The HPLC traces were monitored at 255 nm for cobalamins (a) and at 340 nm for derivatized GSH thioethers (b) as described under “Experimental Procedures.” The reaction mixtures (50:70 μm in both samples) for MMACHC·AdoCbl (upper traces) and MMACHC·MeCbl (middle traces) were prepared in 100 mm HEPES (pH 8.0), 150 mm KCl, and 10% glycerol. The reaction was started with 50 μm GSH and incubated in the dark for 30 min (MMACHC·MeCbl + GSH) or overnight (MMACHC·AdoCbl + GSH) at 20 °C. The products were analyzed by HPLC as described under “Experimental Procedures.” The elution profiles of standards are shown in the lower traces. MeSG, S-methylglutathione.
FIGURE 4.
FIGURE 4.
Dealkylation of MeCbl bound to MMACHC under anaerobic conditions. Addition of anaerobic GSH (50 μm) to an anaerobic solution of MMACHC·MeCbl (70:50 μm) in anaerobic 100 mm HEPES (pH 8.0) containing 150 mm KCl and 10% glycerol at 20 °C resulted in spectral changes consistent with formation of cob(I)alamin. The inset shows the increase in absorbance at 390 nm (○) for cob(I)alamin formation and the single exponential fit to the data (solid line).
FIGURE 5.
FIGURE 5.
Processing of [57Co]MeCbl by normal and cblC mutant human fibroblasts (WG1801, WG2176, and WG3354). Cells were cultured in the presence of 0.125 nm [57Co]MeCbl (0.06 μCi/ml of culture medium) for 48 h. 57Co-Labeled cobalamins were then extracted and analyzed by HPLC as described (2).

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