A method for the production, purification and liposome reconstitution of cobamide synthase.

Abstract

Cobamides are essential for the performance of a variety of reactions such methyl transfers, carbon skeleton rearrangements, and eliminations in both prokaryotes and eukaryotes. However, cobamide biosynthesis is limited to a subset of bacteria and archaea. The biosynthesis pathway culminates with the activation and attachment of a lower ligand to the corrin ring; this branch of the pathway is known as nucleotide loop assembly (NLA) pathway. The cobamide synthase (CobS) enzyme is the penultimate step in NLA pathway, and catalyzes the attachment of an α-ribotide to the activated corrin ring. While other NLA enzymes have been well-studied, studies of CobS have proven difficult to date. CobS is an integral membrane protein, and limitations have been largely due to difficulties in protein purification. Here we provide a method to purify CobS, reconstitute protein in proteoliposomes, and assay for its activity.