Biochemical Approaches for Understanding Iron-Sulfur Cluster Regeneration in Escherichia coli Lipoyl Synthase During Catalysis

Abstract

Lipoyl synthase (LipA in bacteria) is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the second step of the de novo biosynthesis of the lipoyl cofactor: the insertion of sulfur at C6 and C8 of a pendant octanoyl chain. In addition to the [4Fe4S] cluster that is characteristic of the radical SAM (RS) enzymes, LipA contains a second [4Fe4S] cluster that, though controversial, has been proposed to be degraded during turnover to supply the inserted sulfur atoms. A consequence of this proposed role is that the destruction of its iron-sulfur cluster renders the enzyme in an inactive state. Recently, it was shown that Escherichia coli proteins NfuA or IscU can confer catalytic properties to E. coli LipA in vitro. In this chapter, we present methods for characterizing LipA and analyzing its activity in vitro, and provide strategies to monitor the pathway for the regeneration of LipA's auxiliary cluster by E. coli iron-sulfur carrier protein NfuA.

Keywords: Iron–sulfur cluster; Lipoic acid; Protein maturation; Radical; S-Adenosylmethionine.

Figure 1.

SDS-PAGE analysis of (A) E. coli LipA overproduction and (B) E. coli NfuA overproduction. The lanes are as follows: (1) Molecular Weight Ladder (2) Uninduced cells (3) Arabinose-induced cells (4) IPTG-induced cells. The arrows point to the protein of interest.

Figure 2.

SDS-PAGE analysis of (A) E. coli LipA isolation and (B) E. coli NfuA isolation. The lanes are as follows: (1) Molecular Weight Ladder (2) Pellet (3) Supernatant (4) Flow-through (5) Wash 1 (6) Wash 2 (7) Pure protein. The arrows point to the protein of interest.

Figure 3.

UV-visible spectrum of (A) As-isolated E. coli LipA and (B) As-isolated E. coli NfuA.

Figure 4.

(A) Iron Analysis Standard Curve (B) Sulfide Analysis Standard Curve.

Figure 5.

Molecular Sieve Chromatography. (A) Chromatogram of the suite of standards used to generate the standard curve as follows: (A) Blue Dextran, 2000 kDa; (B) β-Amylose, 200 kDa; (C) Alcohol Dehydrogenase, 150 kDa; (D) Bovine Serum Albumin, 66 kDa; (E) Carbonic Anhydrase, 29 kDa; (F) Cytochrome C, 12.4 kDa. (B) Standard curve generated from the elution volume of each of the standards corrected for the void volume of the column versus the log molecular weight of each of the standards.

Figure 6.

Time-dependent formation of the monothiolated intermediate and lipoyl product in the absence (15 μM LipA, shown in black) and presence (400 μM NfuA, shown in red) of NfuA.