IspH Protein ofEscherichiacoli:  Studies on Iron−Sulfur Cluster Implementation and Catalysis

Abstract

The ispH gene of Escherichia coli specifies an enzyme catalyzing the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl diphosphate into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the nonmevalonate isoprenoid biosynthesis pathway. The implementation of a gene cassette directing the overexpression of the isc operon involved in the assembly of iron-sulfur clusters into an Escherichia coli strain engineered for ispH gene expression increased the catalytic activity of IspH protein anaerobically purified from this strain by a factor of at least 200. For maximum catalytic activity, flavodoxin and flavodoxin reductase were required in molar concentrations of 40 and 12 microM, respectively. EPR experiments as well as optical absorbance indicate the presence of a [3Fe-4S](+) cluster in IspH protein. Among 4 cysteines in total, the 36 kDa protein carries 3 absolutely conserved cysteine residues at the amino acid positions 12, 96, and 197. Replacement of any of the conserved cysteine residues reduced the catalytic activity by a factor of more than 70 000.

Methylerythritol phosphate pathway to isoprenoids: Kinetic modeling andin silicoenzyme inhibitions inPlasmodium falciparum

“…The resultant fluxes and all the metabolite concentrations were found to be comparable to the physiological range ( Table 1 and Table 2). At steady state, the ratio at which IPP and DMAPP were synthesized was equal to 6.3:1 as was observed experimentally [27].…”

“…This enzyme catalyzes two reactions, one in which HMBPP gets reduced to IPP, and, the second in which HMBPP gets reduced to DMAPP. The ratio of IPP to DMAPP produced by the enzyme was found to be 6.3:1 [27]. As most of the reductase enzymes work by ordered sequential mechanism [21], this enzyme was also assumed to follow the same mechanism.…”

“…The two equations are similar except that the numerator of Eq. (15) (16) is multiplied with a factor (1/6.3) so that IPP and DMAPP are produced in the ratio observed experimentally [27].…”

Probing the reaction mechanism of IspH protein by x-ray structure analysis

“…The E126Q mutant was prepared as reported earlier (5) using mismatch PCR (15). The recombinant ispH gene was expressed under aerobic conditions in an E. coli XL-1 strain harboring the additional pACYCiscS-fdx plasmid (15) to ensure assembly of a functional iron-sulfur cluster using Terrific Broth (12 g peptone, 24 g yeast extract, 4 mL glycerol, 12 g KH 2 PO 4 and 62 g K 2 HPO 4 in 1 L | water)supplemented with ampicillin (180 mg∕L), chloramphenicol (25 mg∕L), 1 mM cysteine and ferric ammonium citrate (35 mg∕L). Cells were grown over night at 30°C, harvested, washed with degassed saline in a glove box, and stored anaerobically at −20°C.…”

ErpA, an iron–sulfur (Fe–S) protein of the A-type essential for respiratory metabolism in Escherichia coli

“…We made the assumption that the respiration defect was related to alteration of quinone pools. Indeed, quinone derivatives are electron carriers used by both anaerobic and aerobic respiratory chains and are derived from IPP, whose synthesis in bacteria depends on two essential [4Fe-4S] cluster-containing proteins, namely IspG (formally GcpE) and IspH (formally LytB) (38,39). The erpA null mutation (⌬erpA::cat) was introduced into an engineered E. coli strain, which can synthesize IPP both by its natural endogenous pathway and by the eukaryotic MVA-dependent pathway, a pathway that does not employ Fe-S enzymes (40).…”