title: The Streptomyces coelicolor lipoate-protein ligase is a circularly permuted version of the Escherichia coli enzyme composed of discrete interacting domains.
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.
authors: From the Departments of Biochemistry and.
authors: Xinyun
authors: X
authors: Cao
authors: From the Departments of Biochemistry and Microbiology, University of Illinois, Urbana, Illinois 61801 j-cronan@life.uiuc.edu.
authors: John E
authors: JE
authors: Cronan
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D048372:Streptomyces coelicolor / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008063:Thioctic Acid / Q000378:metabolism*
chemical_list: D000249:Adenosine Monophosphate; D008063:Thioctic Acid; D010453:Peptide Synthases; C072322:lipoate-protein ligase
keywords: Acyl Carrier Protein (ACP); Adenylate; Domain; Krebs Cycle; Ligase; Protein Domain; Protein Evolution; Pyruvate Dehydrogenase Complex (PDC); TCA Cycle; Tricarboxylic Acid Cycle
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