Abstract
ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella enterica was overproduced and enriched to approximately 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 (approximately 70% homogeneous) was obtained after treatment with detergent. Under conditions in which cobalamin (Cbl) was saturating, Km(ATP) = 10 microm, kcat = 0.03 s(-1), and Vmax = 54.5 nm min(-1). Similarly, under conditions in which MgATP was saturating, Km(Cbl) = 4.1 microm, 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 > or =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 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.
Highlights
Bioinformatics analysis of the CobA, PduO, and EutT proteins shows that they evolved from different ancestors
Recombinant EutT protein was overproduced in E. coli, but remained insoluble unless cells containing EutT protein were lysed under anoxic conditions using CHES/glycine buffer, followed by resuspension of EutT from the pelleted material with 8 mM CHAPS and a final centrifugation step
EutT activity were erratic when KBH4 was used as reductant to convert cob(III)alamin to cob(I)alamin in vitro
Summary
Strains were grown overnight in 2 ml of lysogenic broth [19, 20] containing the appropriate antibiotic; cultures were incubated at 37 °C. Plates were placed in the chamber of a BioTek plate reader with the temperature held at 37 °C; shaking was constant at a maximum setting
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