Characterization of the Auxiliary Iron-Sulfur of Escherichia Coli Lipoyl Synthase

ABSTRACT Lipoyl Synthase (LipA) catalyzes the last step in the de novo biosynthesis of the lipoyl cofactor, in which two sulfur atoms are installed at C6 and C8 of an octanoyl chain bound covalently in an amide linkage to the ε-amino group of a target lysyl residue of a lipoyl carrier protein (LCP). The enzyme is a member of the radical S-adenosylmethionine (SAM) superfamily, a group of enzymes totaling over 48,000 individual sequences that use a [4Fe–4S] cluster to catalyze a reductive cleavage of SAM to form a 5’-deoxyadenosyl 5’-radical (5’-dA•). This radical plays a key role in each reaction by abstracting a hydrogen atom (H•) from an enzyme-bound substrate. In contrast to most radical SAM (RS) enzymes, LipA contains an additional [4Fe–4S] cluster, termed the auxiliary cluster, that is believed to be the source of the sulfur atoms inserted into the octanoyl chain. Therefore, the enzyme is inactivated after each turnover. Structural studies of LipA from Mycobacterium tuberculosis indicate that the auxiliary cluster is ligated by three cysteine residues and one serine residue. Although cysteine residues are common ligands to [4Fe–4S] clusters, serine ligands have never been observed in [4Fe–4S] clusters of wild-type proteins. Herein, we show that the serine ligand is requisite for Escherichia coli LipA activity. When the serine ligand was altered to a cysteine ligand, the enzyme could insert one sulfur atom into the substrate octanoyl chain, but not the second sulfur atom, even when one of the other three cysteine residues was changed to a serine residue. These experiments suggest that the serine ligand plays an important role in the degradation process of the [4Fe–4S] cluster that allows the enzyme to insert the second sulfur atom.

In a second set of experiments, 2,4-hexadienoic acid, a conjugated six-carbon diene analog of the octanoyl substrate was enzymatically attached to the H protein of the glycine cleavage system, a key cellular LCP. When this substrate analog was incubated with LipA and SAM under turnover conditions, an electron paramagnetic resonance (EPR) spectrum was observed that is consistent with the presence of a delocalized unpaired electron on the substrate analog. Further pulsed EPR techniques show that the radical is magnetically coupled to 57Fe nuclei of the [4Fe-4S]2+ cluster, indicating that it is within 2-3 Å of the cluster. This study presents the first direct evidence for the participation of radical species in radical SAM enzymes that catalyze sulfur insertion, and support the idea that the auxiliary cluster acts as a sulfur source.

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Work Title Characterization of the Auxiliary Iron-Sulfur of Escherichia Coli Lipoyl Synthase
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Penn State
Creators
  1. Justin M. Rectenwald
Keyword
  1. chemistry
  2. Lipoyl Synthase
  3. Escherichia Coli Lipoyl Synthase
  4. iron-sulfur
License Attribution-NonCommercial-NoDerivs 3.0 United States
Work Type Thesis
Acknowledgments
  1. Carsten Krebs (thesis reader)
  2. Squire Booker (research mentor)
Publisher
  1. Eberly College of Science
  2. Department of Chemistry
Publication Date Spring 2014
Subject
  1. Chemistry
Language
  1. English
Deposited November 06, 2014

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