Purification and Initial Characterization of the Salmonella enterica PduO ATP:Cob(I)alamin Adenosyltransferase

Abstract

ABSTRACT The PduO enzyme of Salmonella enterica is an ATP:cob(I)alamin adenosyltransferase that catalyzes the final step in the conversion of vitamin B 12 to coenzyme B 12 . The primary physiological role of this enzyme is to support coenzyme B 12 -dependent 1,2-propanediol degradation, and bioinformatic analysis has indicated that it has two domains. Here the PduO adenosyltransferase was produced in Escherichia coli , solubilized from inclusion bodies, purified to apparent homogeneity, and partially characterized biochemically. The K m values of PduO for ATP and cob(I)alamin were 19.8 and 4.5 μM, respectively, and the enzyme V max was 243 nmol min −1 mg of protein −1 . Further investigations showed that PduO was active with ATP and partially active with deoxy-ATP, but lacked measurable activity with other nucleotides. 31 P nuclear magnetic resonance established that triphosphate was a product of the PduO reaction, and kinetic studies indicated a ternary complex mechanism. A series of truncated versions of the PduO protein were produced in Escherichia coli , partially purified, and used to show that adenosyltransferase activity is associated with the N-terminal domain. The N-terminal domain was purified to near homogeneity and shown to have biochemical properties and kinetic constants similar to those of the full-length enzyme. This indicated that the C-terminal domain was not directly involved in catalysis or substrate binding and may have another role.