How Does NfuA Regenerate a [4Fe-4S] Cluster in Lipoyl Synthase?
Open Access
- Author:
- Pendyala, Jay Venkata
- Area of Honors:
- Biochemistry and Molecular Biology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Amie Kathleen Boal, Thesis Supervisor
Joseph C. Reese, Thesis Honors Advisor - Keywords:
- LipA
NfuA
radical SAM
lipoic acid
iron-sulfur cluster - Abstract:
- Lipoyl synthase (LipA) sacrifices its auxiliary 4Fe-4S cluster to insert two sulfur atoms at carbons C6 and C8 of an octanoyl peptide substrate. This step completes the biosynthesis of lipoic acid, an essential enzyme cofactor for aerobic metabolism. In principle, the sacrificial nature of LipA’s mechanism limits its ability to perform multiple turnovers. However, another iron sulfur carrier protein, NfuA, reassembles LipA’s auxiliary cluster to enable multi-turnover activity in Escherichia coli (Ec). Moreover, the N-terminal domain of Ec NfuA has been shown to be essential for tight interaction with E. coli LipA, thereby facilitating multiple turnovers. NfuA’s conserved C-terminal Fe-S cluster binding motif, CXXC, facilitates the regeneration of the auxiliary Fe-S cluster in LipA. In the human NfuA homolog, NFU1, a glycine to cysteine mutation, G208C, near the CXXC motif is lethal. This mutation impairs lipoic acid synthase (LAS) and the activity of the pyruvate dehydrogenase complex (PDHC). Here, we seek to understand the mechanism of NfuA-mediated cluster transfer via characterization of a thermophilic homolog from Thermosynechococcus elongatus (Te). Unlike Ec NfuA (21 kDa), Te NfuA (12 kDa) doesn’t carry an A-type N-terminal domain. To study the LipA-NfuA interaction via activity and binding assay, we used four T. elongatus proteins including LipA wild-type (wt), NfuA wt, a fusion of Ec NfuA N-term/ Te NfuA, and Te NfuA G55C. We also propose Mӧssbauer spectroscopy and crystallography studies of Te NfuA to address a long-standing gap in knowledge about this important iron-sulfur cofactor chaperone. Achieving these objectives will be useful to studying the properties of clinically relevant variants.