Abstract
The final step in the conversion of vitamin B(12) into coenzyme B(12) (adenosylcobalamin, AdoCbl) is catalyzed by ATP:cob(I)alamin adenosyltransferase (ATR). Prior studies identified the human ATR and showed that defects in its encoding gene underlie cblB methylmalonic aciduria. Here two common polymorphic variants of the ATR that are found in normal individuals are expressed in Escherichia coli, purified, and partially characterized. The specific activities of ATR variants 239K and 239M were 220 and 190 nmol min(-1) mg(-1), and their K(m) values were 6.3 and 6.9 mum for ATP and 1.2 and 1.6 mum for cob(I)alamin, respectively. These values are similar to those obtained for previously studied bacterial ATRs indicating that both human variants have sufficient activity to mediate AdoCbl synthesis in vivo. Investigations also showed that purified recombinant human methionine synthase reductase (MSR) in combination with purified ATR can convert cob(II)alamin to AdoCbl in vitro. In this system, MSR reduced cob(II)alamin to cob(I)alamin that was adenosylated to AdoCbl by ATR. The optimal stoichiometry for this reaction was approximately 4 MSR/ATR and results indicated that MSR and ATR physically interacted in such a way that the highly reactive reaction intermediate [cob(I)alamin] was sequestered. The finding that MSR reduced cob(II)alamin to cob(I)alamin for AdoCbl synthesis (in conjunction with the prior finding that MSR reduced cob(II)alamin for the activation of methionine synthase) indicates a dual physiological role for MSR.
Highlights
The vitamin B12 coenzymes, adenosylcobalamin (AdoCbl)1 and methylcobalamin (CH3Cbl), are required cofactors for at least 15 different enzymes that have a broad but uneven distribution among living forms [1, 2]
Prior studies indicated that cblF mutations affect cobalamin transport from the lysosome to the cytoplasm, whereas cblC and cblD mutations impair the conversion of complex precursors to cob(II)alamin [20, 21]
Because prior studies have shown that the human ATR requires cob(I)alamin for AdoCbl synthesis and is inactive with cob(II)alamin, these results indicate that methionine synthase reductase (MSR) reduced cob(II)alamin to cob(I)alamin for AdoCbl synthesis by ATR
Summary
The vitamin B12 coenzymes, adenosylcobalamin (AdoCbl) and methylcobalamin (CH3Cbl), are required cofactors for at least 15 different enzymes that have a broad but uneven distribution among living forms [1, 2]. The second B12dependent enzyme known in humans is CH3Cbl-dependent methionine synthase (MS). This enzyme is found in the cytoplasm where it catalyzes the conversion of methyltetrahydrofolate and homocysteine to tetrahydrofolate and methionine [6]. In humans, inherited defects in the MCM or MS structural genes or in the genes needed for the synthesis of B12 coenzymes result in methylmalonic aciduria, homocystinuria, or combined disease. These rare disorders, which are often fatal in the first year of life, result from recessive autosomal mutations that fall into nine complementation groups (mut, cblABCDEFGH) [18, 19]. CoA carboxylase; MCEE, methylmalonyl-CoA epimerase; MCM, AdoCbl-dependent methylmalonyl-CoA mutase; CNCbl, vitamin B12; GSCbl, glutathionylcobalamin; THF, tetrahydrofolate
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