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J. Biol. Chem., Vol. 279, Issue 46, 47536-47542, November 12, 2004

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Human ATP:Cob(I)alamin Adenosyltransferase and Its Interaction with Methionine Synthase Reductase^*

Nicole A. Leal, Horatiu Olteanu, Ruma Banerjee, and Thomas A. Bobik¶||

From the Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611, the Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588, and the ^¶Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011

The final step in the conversion of vitamin B₁₂ into coenzyme B₁₂ (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 µM for ATP and 1.2 and 1.6 µM 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 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.

Received for publication, May 17, 2004 , and in revised form, August 24, 2004.

^* This work was supported by National Institutes of Health Grants DK064771 (to T. A. B.) and DK64959 and DK45776 (to R. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed. Tel.: 515-294-4165; Fax: 515-294-0453; E-mail: bobik{at}iastate.edu.

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