The bi-functional enzyme, adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase (CobU), is involved in the biosynthesis of cobalamin in Salmonella typhimurium. and, therefore. can be used for the in vitro synthesis of analogs of B-12. Previously, five different steps were required to purify the recombinant enzyme from Escherichia coli. Here. we describe the cloning, sequencing, and expression of the cobU gene from S. typhimurium ATCC 19585 and, without introducing a purification tag sequence to the N- or C-terminus of the recombinant enzyme, a new single-step purification method based on hydrophobic interaction chromatography. (c) 2005 Elsevier Inc. All rights reserved.