A mechanistic investigation of lipoyl synthase from Escherichia coli and Staphylococcus aureus
Open Access
- Author:
- Rankin, Ananda
- Millennium Scholars Program:
- Microbiology (MICRB)
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisor:
- Squire J Booker, Thesis Supervisor
- Keywords:
- LipA
Lipoyl synthase
Iron Sulfur Cluster
Lipoic Acid
Lipoyl Cofactor
Radical SAM Enzyme - Abstract:
- The lipoyl cofactor plays an integral role in many metabolic processes. The second step of the de novo biosynthesis pathway of the lipoyl cofactor is catalyzed by the radical S-adenosyl-L-methionine (SAM) enzyme, lipoyl synthase (LipA). Radical SAM enzymes contain at least one [4Fe-4S] cluster, which is used to reductively cleave SAM into L-methionine and a 5′-deoxyadenosyl 5’-radical (5′-dA•). LipA contains two [4Fe-4S] clusters; one cluster is used for the SAM cleavage, while the other is sacrificed as a sulfur source for the production of lipoic acid. Two equivalents of 5′-dA• are used to abstract hydrogen atoms from the C6 and C8 positions of an octanoyl-lipoyl carrier protein (LCP) substrate, and hydrogen atoms are replaced with sulfide ions. In vitro studies show that LipA is restricted to only one turnover due to the requirement for the destruction of its auxiliary cluster, but it is currently unknown what enzymes are required to regenerate the catalytic form of this enzyme. Previous studies have implicated Nfu and SufT in the regeneration of the auxiliary cluster of LipA in Staphylococcus aureus. SufT is hypothesized to reconstitute the cluster in S. aureus, as it has been proposed to aid in the maturation of iron-sulfur (Fe-S) proteins. Nfu is an Fe-S cluster carrier protein that has been shown to act synergistically with SufT. In this work, LipA was isolated and shown to be active and to contain two [4Fe-4S] clusters, while Nfu was shown to coordinate one [4Fe-4S] cluster. The activity of Nfu and LipA was monitored using liquid chromatography coupled to mass spectrometry (LC-MS). These results showed that Nfu alone has no effect on the activity of LipA. A sequence alignment of twelve lipA genes revealed that the auxiliary cluster of LipA is coordinated by three cysteine residues in a CX4CX5C motif and a serine residue in a conserved RSSY motif. To determine the importance of each amino acid residue in LipA catalysis, site-directed mutagenesis was used to create single amino acid substitutions. Using activity assays, it was determined that the arginine (R) and two serine (S) residues were necessary for catalytic activity, while the tyrosine (Y) is dispensable. Finally, the lip locus contains two genes, ybeD and ybeF, which have yet to be characterized. As the remainder of the genes in the locus are implicated in lipoic acid biosynthesis, it was hypothesized the two proteins were involved with lipoic acid biosynthesis. In order to study them, ybeD was overexpressed and purified, and LipA activity assays were performed in the presence of YbeD. The addition of YbeD to the reaction had no effect on LipA catalysis.