The Streptomyces coelicolor Lipoate-Protein Ligase is a Circularly Permuted Version of the Escherichia coli Enzyme Composed of Discrete Interacting Domains [Microbiology]

January 27th, 2015 by Cao, X., Cronan, J. E.

Lipoate-protein ligases are used to scavenge lipoic acid from the environment and attach the coenzyme to its cognate proteins which are generally the E2 components of the 2-oxoacid dehydrogenases. The enzyme uses ATP to activate lipoate to its adenylate, lipoyl-AMP, which remains tightly bound in the active site. This mixed anhydride is attacked by the ɛ-amino group of a specific lysine present on a highly conserved acceptor protein domain resulting in the amide-linked coenzyme. The Streptomyces coelicolor genome encodes only a single putative lipoate ligase. However, this protein had only low sequence identity (<20%) to the lipoate ligases of demonstrated activity and appeared to be a circularly permuted version of the known lipoate ligase proteins in that the canonical C-terminal domain seemed to have been transposed to the N-terminus. We have tested the activity of this protein both by in vivo complementation of an Escherichia coli ligase-deficient strain and by in vitro assays. Moreover, when the domains are rearranged into a protein that mimicked the arrangement found in the canonical lipoate ligases, the enzyme retained complementation activity. Finally when the two domains were separated into two proteins, both domain proteins were required for complementation and for catalysis of the overall ligase reaction in vitro. However, only the large domain protein was required for transfer of lipoate from the lipoyl-AMP intermediate to the acceptor proteins whereas both domain proteins were required to form lipoyl-AMP.
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