Biochemistry, Department of


Date of this Version



The Journal of Biological Chemistry, VOL. 281, NO. 25, pp. 16971–16977, June 23, 2006


Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc.


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 >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 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.