The Streptomyces coelicolor Lipoate-protein Ligase Is a Circularly Permuted Version of the Escherichia coli Enzyme Composed of Discrete Interacting Domains |
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Authors: | Xinyun Cao John E Cronan |
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Institution: | From the Departments of ‡Biochemistry and ;§Microbiology, University of Illinois, Urbana, Illinois 61801 |
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Abstract: | 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 enzymes use ATP to activate lipoate to its adenylate, lipoyl-AMP, which remains tightly bound in the active site. This mixed anhydride is attacked by the ϵ-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 (<25%) to the lipoate ligases of demonstrated activity and appears to be a circularly permuted version of the known lipoate ligase proteins in that the canonical C-terminal domain seems to have been transposed to the N terminus. We 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 were 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-containing proteins were required for complementation and catalysis of the overall ligase reaction in vitro. However, only the large domain-containing protein was required for transfer of lipoate from the lipoyl-AMP intermediate to the acceptor proteins, whereas both domain-containing proteins were required to form lipoyl-AMP. |
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Keywords: | Acyl Carrier Protein (ACP) Protein Domain Protein Evolution Pyruvate Dehydrogenase Complex (PDC) Tricarboxylic Acid Cycle TCA Cycle Krebs Cycle Adenylate Domain Ligase |
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