title: Purification and initial characterization of the Salmonella enterica PduO ATP:Cob(I)alamin adenosyltransferase.
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 microM, 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.
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, USA.
authors.forename: Celeste L V
authors.initials: CL
authors.lastname: Johnson
authors.forename: Marian L
authors.initials: ML
authors.lastname: Buszko
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Bobik
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D010710:Phosphates / Q000494:pharmacology; D019779:Salmonella enterica / Q000201:enzymology*
chemical_list: D010710:Phosphates; D000255:Adenosine Triphosphate; D019883:Alkyl and Aryl Transferases; C019920:cob(I)alamin adenosyltransferase