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
MgATPwas saturating,Km(Cbl) = 4.1µM, kcat = 0.06 s-1, andVmax=
105 nM min-1. Unlike other ATP:co(I)rrinoid adenosyltransferases
in the cell (i.e. CobA and PduO), EutT activity was \u3e50-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 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
