The Role of the Auxiliary Iron-sulfur Cluster of Lipoyl Synthase in the Biosynthesis of the Lipoyl Cofactor
- Lanz, Nicholas
- [University Park, Pennsylvania] : Pennsylvania State University, 2015.
- Physical Description:
- 1 electronic document
- Additional Creators:
- Booker, Squire J.
- Restrictions on Access:
- Open Access.
- Lipoic acid is an essential cofactor found in all domains of life. It is a simple cofactor composed of an eight-carbon fatty acid chain with sulfur atoms inserted at C6 and C8. The de novo biosynthesis of lipoic acid consists of two dedicated steps. In the first step, an octanoyl transferase, LipB, transfers an octanoyl chain from an acyl-carrier protein to a lipoyl carrier protein (LCP). The octanoyl-LCP serves as the substrate for the second enzyme, lipoyl synthase (LipA). LipA belongs to a superfamily known as radical SAM (RS) enzymes that use a reduced [4Fe--4S] cluster to reductively cleave S-adenosyl-L-methionine (SAM) to methionine and a potent 5'-deoxyadenosyl-5'-radical (5'-dA ). LipA uses two equivalents of 5'-dA to abstract hydrogen atoms from C6 and C8 of the octanoyl chain in a stepwise manner, cleaving two unactivated C--H bonds of the substrate. In addition to the [4Fe--4S] cluster required for radical chemistry, LipA contains an auxiliary [4Fe--4S] cluster that is used as the source of sulfide for the reaction. Radical recombination of the substrate with two of the bridging [mu]3-sulfido ions of the auxiliary cluster results in the intact lipoyl cofactor.Isolation and characterization of the monothiolated intermediate conclusively determined that a covalent cross-link is formed between the octanoyl group of the substrate protein or peptide and the auxiliary iron--sulfur cluster of LipA. This intermediate is shown to be catalytically and kinetically competent to form product. A detailed spectroscopic analysis using Mössbauer spectroscopy revealed that the formation of the cross-linked intermediate corresponds to the loss of an iron ion from the auxiliary cluster, leaving a 3Fe species attached to the substrate. When a conjugated substrate analogue, an LCP with a 2,4-hexadienoyl moiety attached to the appropriate lysine, is used, a substrate radical can be trapped and observed by electron paramagnetic resonance (EPR) spectroscopy. This substrate was reacted with 57Fe-labeled LipA, and hyperfine sublevel correlation spectroscopy, a pulsed EPR technique, was employed. The substrate radical was shown to interact with the auxiliary iron--sulfur cluster of LipA, and the distance of the radical was mapped to within 2 Å of the cluster, positioning the substrate to react with the sulfide ions of the cluster.An analysis of the LipA from Mycobacterium tuberculosis (MtLipA) showed that this enzyme contains the same cluster species as found in the enzyme from Escherichia coli (EcLipA). The reaction of MtLipA proceeds through the same intermediate with similar kinetics. MtLipA can also complement lipA auxotrophic mutants of E. coli, demonstrating the similarity of the two enzymes. However, the oligomeric state of MtLipA differs from that of EcLipA. EcLipA exists as a mixture of monomers and dimmers that can vary based on the buffer solution and presence or absence of SAM. MtLipA, on the other hand, exists solely as a monomer under the conditions tested. It was also found that the substrate specificity of LipA can be reduced to just the octanoyllysine moiety.Finally, recently solved X-ray crystal structures of LipA provided a hint as to the role of a conserved RSSY motif found in the C-terminal tail of LipA. The arginine of this motif was found to be critical for binding of the substrate to LipA. The second serine of the motif acts as a ligand to the fourth iron ion of the auxiliary cluster. Activity of LipA is greatly diminished when this residue is substituted with an alanine or a phenylalanine. Furthermore, when the serine ligand is substituted with a cysteine, the enzyme is capable of generating a monothiolated intermediate, but cannot form the lipoyl product. Instead, an aberrant unsaturated fatty acyl product is formed. A spectroscopic analysis of the intermediate formed by the Ser to Cys variant showed that the 3Fe species is not generated by this enzyme, and a high-spin [4Fe--4S] cluster is found in its place.
- Dissertation Note:
- Ph.D. Pennsylvania State University, 2015.
- Reproduction Note:
- Microfilm (positive). 1 reel ; 35 mm. (University Microfilms 107-59915)
- Technical Details:
- The full text of the dissertation is available as an Adobe Acrobat .pdf file ; Adobe Acrobat Reader required to view the file.
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