Purification and initial biochemical characterization of ATP:Cob(I)alamin adenosyltransferase (EutT) enzyme of Salmonella enterica

26Citations
Citations of this article
26Readers
Mendeley users who have this article in their library.

This article is free to access.

Abstract

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 β-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 PP i 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. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Cite

CITATION STYLE

APA

Buan, N. R., & Escalante-Semerena, J. C. (2006). Purification and initial biochemical characterization of ATP:Cob(I)alamin adenosyltransferase (EutT) enzyme of Salmonella enterica. Journal of Biological Chemistry, 281(25), 16971–16977. https://doi.org/10.1074/jbc.M603069200

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Save time finding and organizing research with Mendeley

Sign up for free