Structure and Function of Four Classes of the 4Fe–4S Protein, IspH

Abstract

IspH, E-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate reductase, is an essential enzyme in isoprenoid biosynthesis and an important drug/herbicide target. Using X-ray crystallographic, bioinformatics, mutagenesis/kinetics/stability and EPR results we show that organisms from different environments ultilize one of four main IspH classes. The classes are based on the arrangement of the aromatic residues near the 4Fe-4S cluster and the presence or absence of N- and C-terminal extensions. They are: Class A, found primarily in anaerobic/microaerophilic bacteria; Class B, found in aerobic bacteria; Class C, found in cyanobacteria/plants; and Class D, found in apicomplexan parasites. Using mutagenesis, we show that the cluster-associated aromatic groups in Class A and a Class B IspHs enhance cluster oxidative stability. Y198A, F302A and a C-terminus truncation mutant of the Class B (E. coli) IspH have lower catalytic activity than wild-type protein when using methyl viologen as electron donor, but higher activity with dithionite as electron donor, due to ready access of the small reductant to the cluster, consistent with their increased oxygen/H2O2 sensitivity. F302A has the largest effect on the reaction rates and EPR studies indicate this residue affects Fe-S cluster structure. Similar effects on cluster stability are seen with Class A (F14A and Y98A) mutants, but effects on ET rates are smaller and there are no differences between the EPR spectra of mutant and wild-type proteins. Overall, the results are of general interest since they show, for the first time, that there are multiple IspH classes that have evolved to enable organisms to survive in diverse oxidative-stress environments.

Systems-Level Analysis of Oxygen Exposure in Zymomonas mobilis : Implications for Isoprenoid Production

“…The metabolic bottleneck in the lower MEP pathway was likely caused by oxidative damage to the [4Fe-4S] cluster cofactors of IspG and IspH; the sensitivity of IspG and IspH to oxidative stress is well documented in bacteria and in plants as well as with purified enzyme (20–23). To confirm this, we exposed Z.…”

Genomic Enzymology: Web Tools for Leveraging Protein Family Sequence–Function Space and Genome Context to Discover Novel Functions

“…40 (E) SSN for IspH in isoprenoid biosynthesis. 56 (F) SSNs for members of the DRE-TIM metallolyase superfamily. 52 Figures reproduced with permission from refs (40), (43), (52), (56), (60), and (76).…”

Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non‐diphosphate Inhibitors

“…This structurei ss urprisingly labile,l eading to loss of the fourth Fe under crystallization conditions. Ligand-free IspH is also very sensitivet oO 2 ,w hereas HMBPP and PPP,a sw ell as an enolateliganded species, are much less sensitivet oc luster degradation by O 2, [17] suggesting that the presence of relatively bulky ligand side chains might block the H 2 O/O 2 binding that leads to lability.F or example, in the PPP (5)s tructure (crystallized from oxidized IspH), there is as ingle H 2 O( or OH À )b ound to the fourth Fe, [14] whereas the acetylene group is % 3.6 from the Fe, acting perhaps as ab arriert ow ater and oxygen ligands.…”