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Originally published In Press as doi:10.1074/jbc.M603069200 on April 24, 2006

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*Formula

Nicole R. Buan1 and Jorge C. Escalante-Semerena2

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, Km(ATP) = 10 µM, kcat = 0.03 s–1, and Vmax = 54.5 nM min–1. Similarly, under conditions in which MgATP was saturating, Km(Cbl) = 4.1 µM, kcat = 0.06 s–1, and Vmax = 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 beta-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 (PPPi). Results from 31P NMR spectroscopy studies identified PPi and Pi as by-products of the EutT reaction. In the absence of Cbl, EutT cleaved ATP into adenosine and PPPi, suggesting that PPPi is broken down into PPi and Pi. 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.

Formula 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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M. St. Maurice, P. E. Mera, M. P. Taranto, F. Sesma, J. C. Escalante-Semerena, and I. Rayment
Structural Characterization of the Active Site of the PduO-Type ATP:Co(I)rrinoid Adenosyltransferase from Lactobacillus reuteri
J. Biol. Chem., January 26, 2007; 282(4): 2596 - 2605.
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