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

ATP:cob(I) alamin adenosyltransferase (EutT) of Salmonella enterica was overproduced and enriched to similar 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 ( similar to 70% homogeneous) was obtained after treatment with detergent. Under conditions in which cobalamin (Cbl) was saturating, K-m(ATP) = 10 mu M, 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 mu M, 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 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 P-31 NMR spectroscopy studies identified PPi and P-i 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 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.