Expression, purification, and physical characterization of Escherichia coli lipoyl(octanoyl)transferase

Natasha M. Nesbitt, Camelia Baleanu-Gogonea, Robert M. Cicchillo, Kathy Goodson, David F. Iwig, John A. Broadwater, Jeffrey A. Haas, Brian G. Fox, Squire J. Booker

Research output: Contribution to journalArticlepeer-review

27 Scopus citations

Abstract

Lipoic acid is a sulfur-containing 8-carbon fatty acid that functions as a central cofactor in multienzyme complexes that are involved in the oxidative decarboxylation of glycine and several α-keto acids. In its functional form, it is bound covalently in an amide linkage to the ε-amino group of a conserved lysine residue of the "lipoyl bearing subunit," resulting in a long "swinging arm" that shuttles intermediates among the requisite active sites. In Escherichia coli and many other organisms, the lipoyl cofactor can be synthesized endogenously. The 8-carbon fatty-acyl chain is constructed via the type II fatty acid biosynthetic pathway as an appendage to the acyl carrier protein (ACP). Lipoyl(octanoyl)transferase (LipB) transfers the octanoyl chain from ACP to the target lysine acceptor, generating the substrate for lipoyl synthase (LS), which subsequently catalyzes insertion of both sulfur atoms into the C-6 and C-8 positions of the octanoyl chain. In this study, we present a three-step isolation procedure that results in a 14-fold purification of LipB to >95% homogeneity in an overall yield of 25%. We also show that the protein catalyzes the transfer of the octanoyl group from octanoyl-ACP to apo-H protein, which is the lipoyl bearing subunit of the glycine cleavage system. The specific activity of the purified protein is 0.541 U mg -1, indicating a turnover number of ∼0.2 s -1, and the apparent K m values for octanoyl-ACP and apo-H protein are 10.2 ± 4.4 and 13.2 ± 2.9 μM, respectively.

Original languageEnglish (US)
Pages (from-to)269-282
Number of pages14
JournalProtein Expression and Purification
Volume39
Issue number2
DOIs
StatePublished - Feb 2005

All Science Journal Classification (ASJC) codes

  • Biotechnology

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