43432-G4
Hydrogen Abstraction from an Unactivated Hydrocarbon Chain: Structure and Function of Lipoate Synthase
We are trying to understand the chemical reaction catalyzed by lipoate synthase (LipS). This protein employs powerful radical chemistry to modify a hydrocarbon substrate. The key to the protein's reactivity lies in the cube-like 4Fe-4S iron-sulfur clusters which are thought to form part of the protein's active site. The iron-sulfur clusters can reductively cleave S-adenosylmethionine (SAM), generating adenosyl radical which is used to abstract hydrogen atom from an octanoyl group on the substrate. We would like to see how the LipS protein arranges the two 4Fe-4S clusters relative to S- SAM, and the substrate. Our ultimate goal is to determine the structure of LipS to understand how the protein generates and controls radical species during catalysis.
The key limitation in pursuing structural studies is the production of LipS protein with intact iron-sulfur clusters which can be improperly formed, or damaged by exposure to oxygen. We are using a variety of approaches to address this problem. We are using the lipoate synthase protein from P. falciparum and have shown that the iron-sulfur clusters in this protein are less sensitive to oxidative damage than those from other lipoate synthase proteins. We produced LipS in bacterial culture, purified it and showed that it has catalytic activity, however, the iron content of recombinant LipS was about 60% of what we anticipated. We addressed this problem by cotransformed the LipS expressing E. coli with a second plasmid (pDB1282 from John Dean) that augments the iron sulfur cluster assembly machinery found in E. coli. We also changed our purification scheme to avoid using polyhistidine tags as a purification tool since the metal-chelate affinity resins tend to remove iron from LipS. Lastly, we generated a new strain of E. coli designed to prevent damage to the iron-sulfur clusters in vivo. This strain lacks the two E. coli enzymes that are capable of producing octanoylated substrates for LipS. Without these substrates, LipS should not undergo catalysis, a process shown to degrade the iron-sulfur clusters.
In spite of the approaches described above, we still had a hard time purifying recombinant LipS. The protein always seemed to copurify with several persistent proteins even when alternative purification strategies were used. Through amino acid sequencing, we were able to identify these proteins and found that they constitute dehydrogenase complexes that are known to be lipoylated in E. coli. This was an unexpected result since the E.coli dehydrogenases are not the native substrates for the malaria lipoate synthase. We experimented with this phenomenon and found that LipS binds to the lipoylation domain of these complexes and that these domains can be in the unmodified apo form. We are now exploiting these results by producing a complex of LipS with a cognate lipoylation domain. The complex serves to shield LipS from degradation and it may also allow us to probe the structural determinants of substrate bidning.
We combined the approaches described above to produce high yields of homogeneous LipS protein for use in structural and functional studies. These studies will show how this protein produces and controls the highly reactive radical species required to catalyze the lipoate synthase reaction.
