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

Guodong Rao, Eric Oldfield

Research output: Contribution to journalArticlepeer-review


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 electron paramagnetic resonance (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. Class A enzymes are found primarily in anaerobic and microaerophilic bacteria. Class B enzymes are found in aerobic bacteria. Class C enzymes are found in cyanobacteria and plants. Class D enzymes are found in apicomplexan parasites. Using mutagenesis, we show that the cluster-associated aromatic groups in class A and class B IspHs enhance cluster oxidative stability. Y198A, F302A, and a C-terminal truncation mutant of the class B (Escherichia coli) IspH have catalytic activity lower than that of the wild-type protein when using methyl viologen as the electron donor, but higher activity with dithionite as the electron donor, due to ready access of the small reductant to the cluster, consistent with their increased oxygen and 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; however, 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 because they show, for the first time, that there are multiple IspH classes that have evolved to allow organisms to survive in diverse oxidative-stress environments.

Original languageEnglish (US)
Pages (from-to)4119-4129
Number of pages11
Issue number29
StatePublished - Jul 26 2016

ASJC Scopus subject areas

  • Biochemistry


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