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 ∼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 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 (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.