Two Iron-Sulfur Proteins Involved in Isoprenoid Biosynthesis with New Catalytic Roles

Abstract

(E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE or IspG) and (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB or IspH) are involved in the last two steps of the DOXP pathway for isoprenoid biosynthesis. GcpE converts 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) in the penultimate step of the DOXP pathway. LytB catalyzes the conversion of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) into two products: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the terminal step of the DOXP pathway. GcpE and LytB are iron-sulfur proteins containing a [4Fe-4S] cluster at the active site. The spectroscopic characterizations of the [4Fe-4S] cluster have been studied. In order to develop potential inhibitors for drugs, the catalytic mechanisms of GcpE and LytB have been investigated. For GcpE, two individual electron transfer steps have different midpoint potentials. The reaction intermediate species have been trapped and studied with titration experiments. The EPR properties of species FeSA are similar to that detected in feredoxin:thioredoxin reductase (FTR), indicating the direct binding from substrate or the reaction intermediate. The characterization of FeSA species has been performed with 57Fe-EPR and ENDOR spectroscopy. The diphosphate group does not directly bind to iron-sulfur cluster based on the distance calculated from the 31P superhyperfine coupling. The FeSB species has been identified as the product binding to the 4Fe cluster. Based on these information, a new mechanism has been proposed for GcpE. For LytB, it is determined that the active form is a [4Fe-4S] cluster and the enzyme activity is related to the cluster content of [4Fe-4S] clusters. A similar transient paramagnetic species FeSI to FeSA species in case of GcpE has been detected after incubation of one-electron-reduced enzyme with substrate. The characterization of FeSI species has been carried out with 57Fe-EPR and ENDOR spectroscopy. It is implied that the diphosphate group does not bind to the iron-sulfur cluster, but the hydroxyl group might be the binding group. The preliminary data from site-directed mutagenesis of LytB (including H42A, H42F, H124A, H124F, E126A and E126Q) identified several mutations that have different effects on iron-sulfur cluster or reaction intermediate. The further exploration will be executed to achieve more details and information about catalytic mechanism. A [4Fe-4S] cluster is discovered at the active site of both GcpE and LytB. After incubation with reductant and substrate, new paramagnetic species are detected during the reaction. Our data indicate that these new paramagnetic species are iron-sulfur based and might be bound by substrate through the hydroxyl group. The mutant studies on LytB suggest that His42, His124 and Glu126 play important roles in the catalysis. The mechanisms for GcpE and LytB are proposed, however, further investigation is necessary for full understanding of them.