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J Biol Chem. 2015 Mar 13;290(11):7280-90. doi: 10.1074/jbc.M114.626879. Epub 2015 Jan 27.

The Streptomyces coelicolor lipoate-protein ligase is a circularly permuted version of the Escherichia coli enzyme composed of discrete interacting domains.

Author information

1
From the Departments of Biochemistry and.
2
From the Departments of Biochemistry and Microbiology, University of Illinois, Urbana, Illinois 61801 j-cronan@life.uiuc.edu.

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.

KEYWORDS:

Acyl Carrier Protein (ACP); Adenylate; Domain; Krebs Cycle; Ligase; Protein Domain; Protein Evolution; Pyruvate Dehydrogenase Complex (PDC); TCA Cycle; Tricarboxylic Acid Cycle

PMID:
25631049
PMCID:
PMC4358146
DOI:
10.1074/jbc.M114.626879
[Indexed for MEDLINE]
Free PMC Article

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