This Is AuburnElectronic Theses and Dissertations

Isoprenoid synthesis: New roles for iron sulfur clusters




Adedeji, Dolapo

Type of Degree



Chemistry and Biochemistry


Spectroscopic investigations of the GcpE and LytB enzymes involved in the last two steps of the DOXP pathway for isoprene synthesis have been described. YfgB, one of the accessory proteins has also been characterized. The LytB enzyme converts (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the last step of the DOXP pathway for isoprenoid synthesis. EPR measurements on the LytB enzyme showed that the purified enzyme contains mixture of [3Fe-4S]+1 and [4Fe-4S]+1 clusters after reduction with dithionite solution. We propose that the LytB enzyme is also a 4Fe cluster containing enzyme. The formation of 3Fe clusters in the active site is due to instability of the 4Fe cluster. The GcpE enzyme converts 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) to (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the second to the last step of the DOXP pathway. Purification of the enzyme under exclusion of molecular oxygen yielded a protein that solely contained a [4Fe-4S] cluster. An unusual paramagnetic species that resembles the signal of a HiPIP-type 4Fe cluster is detected under turnover conditions in the presence of substrate and the artificial reductant dithionite. The EPR signal is similar to that found in ferredoxin:thioredoxin reductase. Since it was shown for the latter enzyme that the 4Fe cluster is directly involved in the binding of substrate, we propose that the 4Fe cluster in GcpE has a similar function. Pre-steady-state experiments showed that the HiPIP-like signal represents a true reaction intermediate. Further characterization of the HiPIP-like signal by Electron nuclear double resonance (ENDOR) spectroscopy revealed the presence of a weak 31P superhyperfine coupling due to the phosphate groups present in the substrate MEcPP. This would further confirm the binding of the substrate to the cluster during the reaction mechanism. However, due to the presence of other EPR-active species this is not a solid conclusion. The YfgB enzyme, one of the accessory proteins of DOXP pathway, belongs to the radical SAM family enzyme. The YfgB enzyme contains a well defined CxxxCxxC motif that is common to all radical SAM enzymes. Reconstitution of the cluster in YfgB resulted in the presence of a [4Fe-4S]2+/1+ cluster. The presence of only one 4Fe cluster would be in line with the role of activating enzyme as in pyruvate formate-lyase activating enzyme (PFL-AE) and anaerobic ribonucleotide reductase activating enzyme (ARR-AE).