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J. Biol. Chem., Vol. 281, Issue 25, 16971-16977, June 23, 2006

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Purification and Initial Biochemical Characterization of ATP:Cob(I)alamin Adenosyltransferase (EutT) Enzyme of Salmonella enterica^*

From the Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53726-4087

ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella enterica was overproduced and enriched to 70% homogeneity, and its basic kinetic parameters were determined. Abundant amounts of EutT protein were produced, but all of it remained insoluble. Soluble active EutT protein (70% homogeneous) was obtained after treatment with detergent. Under conditions in which cobalamin (Cbl) was saturating, K_m(ATP) = 10 µM, k_cat = 0.03 s^–1, and V_max = 54.5 nM min^–1. Similarly, under conditions in which MgATP was saturating, K_m(Cbl) = 4.1 µM, k_cat = 0.06 s^–1, and V_max = 105 nM min^–1. Unlike other ATP:co(I)rrinoid adenosyltransferases in the cell (i.e. CobA and PduO), EutT activity was 50-fold higher with ATP versus GTP, and EutT retained 80% of its activity with ADP substituted for ATP and was completely inactive with AMP as substrate, indicating that the enzyme requires the -phosphate group of the nucleotide substrate. The data suggest that the amino group of adenine might play a role in nucleotide recognition and/or binding. Unlike the housekeeping CobA enzyme, EutT was not inhibited by inorganic tripolyphosphate (PPP_i). Results from ³¹P NMR spectroscopy studies identified PP_i and P_i as by-products of the EutT reaction. In the absence of Cbl, EutT cleaved ATP into adenosine and PPP_i, suggesting that PPP_i is broken down into PP_i and P_i. Electron transfer protein partners for EutT were not encoded by the eut operon. EutT-dependent activity was detected in cell-free extracts of cobA strains enriched for EutT when FMN and NADH were used to reduce cob(III)alamin to cob(I)alamin.

Received for publication, March 31, 2006 , and in revised form, April 20, 2006.

^* This work was supported by Grant RO1 GM40313 from the National Institutes of Health (to J. C. E.-S.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1s.

¹ Howard Hughes Predoctoral Fellow. Present address: Dept. of Microbiology, University of Illinois, Urbana, IL 61801.

² To whom correspondence should be addressed: Dept. of Bacteriology, University of Wisconsin, 1710 University Ave., Madison, WI 53726-4087. Tel.: 608-262-7379; Fax: 608-265-7909; E-mail: escalante{at}bact.wisc.edu.

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