Mechanistic Investigation of the Escherichia coli Lipoyl Synthase

Open Access
Billgren, Elizabeth Star
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Master of Science
Document Type:
Master Thesis
Date of Defense:
October 31, 2008
Committee Members:
  • Squire J Booker, Thesis Advisor
  • glycine cleavage system
  • H protein
  • lipoamidase
  • lipoate protein ligase A
  • lipoyl synthase
  • lipoic acid
  • radical SAM superfamily
The lipoyl cofactor is an essential cofactor employed in various multi-enzyme complexes that have roles in primary metabolism. Each of these complexes contains a lipoyl carrier protein (LCP) to which lipoic acid is tethered via an amide linkage to a conserved lysine residue. In the glycine cleavage system, the LCP is H protein. There are two ways by which the lipoyl cofactor can be synthesized by E. coli – free lipoic acid may be directly attached to a LCP by lipoate protein ligase A (LplA), or the sulfur atoms can be directly inserted into C6 and C8 of a fatty n-octanoyl chain attached to the LCP by lipoyl synthase (LipA). The way in which LipA inserts sulfur atoms into unactivated, saturated carbons has been the subject of many recent biochemical studies. It was determined that LipA contains two [4Fe-4S]2+ clusters essential in catalysis, each of which are ligated by three cysteines in two conserved motifs. The first, harbored in a CX3CX2C motif, is found in members of the radical S-adenosyl-L-methioine (SAM) superfamily of enzymes and is responsible for donating an electron into the sulfonium of SAM yielding highly reactive 5’deoxyadenosyl radicals. In the case of LipA, two of these radicals are thought to abstract hydrogens from C6 and C8. The second motif, CX4CX5C, is conserved only among lipoyl synthases and the [4Fe-4S] cluster housed therein is proposed to be the source of sulfur in the lipoyl product. In support of this role are findings that both product sulfur atoms are sourced by LipA itself and both are donated by a single LipA polypeptide. The proposed mechanism of catalysis by LipA thus invokes carbon-centered substrate radicals at C6 and C8. Direct evidence of these radicals has not yet been presented. In an effort to spectroscopically observe these radicals, a number of substrates for LipA where synthesized with side chains capable of radical stabilization. These side chains were attached to the H protein LCP with LplA and used in reactions with LipA. When a 2,4 hexadienoyl-H protein substrate was used, an organic radical was readily observed by electron paramagnetic resonance (EPR) spectroscopy supporting that carbon-centered radicals are on the LipA reaction pathway. Additionally, if an Fe-S cluster is indeed the source of both sulfur atoms in the lipoyl product, free monothiolated intermediates should not be observed on the reaction pathway, but instead associated with LipA via a carbon-sulfur linkage to the Fe-S cluster. In support of this, a crosslinked species has been identified and isolated containing LipA and an 8,8,8-2H3-octanoyl-H protein. It is known that sulfur insertion at C6 precedes insertion at C8 and that there is a significant deuterium isotope effect at C8, making it likely that LipA and H-protein are crosslinked directly via a sulfur atom in one of the [4Fe-4S] clusters. This species has been characterized by UV-vis, EPR and Mössbauer spectroscopies. The unique signature produced is typical of a [3Fe-4S]0 cluster, which is a possible intermediate on the reaction pathway. Iron and sulfide release have also been observed during LipA catalysis supporting that the cluster degrades following product sulfur donation.