Dataset Information

Mechanistic insights into the reductive dehydroxylation pathway for the biosynthesis of isoprenoids promoted by the IspH enzyme.

ABSTRACT: Here, we report an integrated quantum mechanics/molecular mechanics (QM/MM) study of the bio-organometallic reaction pathway of the 2H+/2e- reduction of (E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate (HMBPP) into the so called universal terpenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), promoted by the IspH enzyme. Our results support the viability of the bio-organometallic pathway through rotation of the OH group of HMBPP away from the [Fe4S4] cluster at the core of the catalytic site, to become engaged in a H-bond with Glu126. This rotation is synchronous with ?-coordination of the C2 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 C3 double bond of HMBPP to the apical Fe atom of the [Fe4S4] cluster. Dehydroxylation of HMBPP is triggered by a proton transfer from Glu126 to the OH group of HMBPP. The reaction pathway is completed by competitive proton transfer from the terminal phosphate group to the C2 or C4 atom of HMBPP.

SUBMITTER: Abdel-Azeim S

PROVIDER: S-EPMC5511988 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Mechanistic insights into the reductive dehydroxylation pathway for the biosynthesis of isoprenoids promoted by the IspH enzyme.

Abdel-Azeim Safwat S Jedidi Abdesslem A Eppinger Jorg J Cavallo Luigi L

Chemical science 20150622 10

Here, we report an integrated quantum mechanics/molecular mechanics (QM/MM) study of the bio-organometallic reaction pathway of the 2H+/2e- reduction of (E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate (HMBPP) into the so called universal terpenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), promoted by the IspH enzyme. Our results support the viability of the bio-organometallic pathway through rotation of the OH group of HMBPP a ...[more]

PMID: 28757951

Similar Datasets

Project description:Purpose: To understand the dynamic transcriptional response of Escherichia coli cells to intracellular HMBPP accumulation. Methods: RNA-seq libraries were constructed for three samples, including (I) CAR005, which used as control; (II) IspG1, which strong over-expressed ispG in CAR005; (III) G4H14, which simultaneously over-expressed ispG and ispH in CAR005.For preparation of RNA samples, single colonies were inoculated into 15x100 mm tubes containing 4 ml LB, and grown at 30ºC and 250 rpm overnight. 100 μl seed culture was inoculated into a 100 ml flask containing 10 ml LB medium, and grown at 30ºC and 250 rpm. After 5 h, 1 ml culture was harvested by centrifugation, frozen in liquid nitrogen and sent to Beijing Genomics Institute (BGI, Shenzhen, China). The three 90-nt paired-end RNA-seq libraries were generated commercially at Beijing Genomics Institute by using the HiSeq™ 2000 platform. Sequencing data was handled essentially with Bowtie2, Tophat2, NOIseq. Expression levels are presented as Reads Per Kilobase of transcript per Million mapped reads (RPKM). Results: We assessed expression levels of a 5 hour culture of ispG1 and compared to the parent strain CAR005. Among the 4200 predicted genes in E. coli ATCC 8739, relative expression levels for 504 genes were found changed in strain ispG1, with 166 markedly up-regulated and 338 repressed. FunCat was used to analyze the biological processes represented by these genes and found categories related to “nucleotide/nucleoside/nucleobase metabolism” (P=7.76E-05), “ribosome biogenesis,” (P=1.06E-20), “translation,” (P=4.26E-12) and “nucleobase binding” (P=1.86E-06) were markedly enriched in the repressed genes set, suggested that HMBPP accumulation might exert deterious effect on cell growth and viability. As for G4H14, only 64 genes were found down-regulated in G4H14. Biological processes associated with macromolecular synthesis no longer markedly enriched, suggesting that cell growth and viability tended to be restored to control level after eliminating HMBPP accumulation. Conclusions: Transcriptome profiling exhibited additional evidence to the cytotoxicity of HMBPP, meanwhile, demonstrated that activating downstream IspH could contribute to attenuation of such deterious effects.

Project description:The recently discovered methylerythritol phosphate (MEP) pathway provides new targets for the development of antibacterial and antimalarial drugs. In the final step of the MEP pathway, the [4Fe-4S] IspH protein catalyzes the 2e(-)/2H(+) reductive dehydroxylation of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) to afford the isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Recent experiments have attempted to elucidate the IspH catalytic mechanism to drive inhibitor development. Two competing mechanisms have recently emerged, differentiated by their proposed HMBPP binding modes upon 1e(-) reduction of the [4Fe-4S] cluster: (1) a Birch reduction mechanism, in which HMBPP remains bound to the [4Fe-4S] cluster through its terminal C4-OH group (ROH-bound) until the -OH is cleaved as water; and (2) an organometallic mechanism, in which the C4-OH group rotates away from the [4Fe-4S] cluster, allowing the HMBPP olefin group to form a metallacycle complex with the apical iron (?(2)-bound). We perform broken-symmetry density functional theory computations to assess the energies and reduction potentials associated with the ROH- and ?(2)-bound states implicated by these competing mechanisms. Reduction potentials obtained for ROH-bound states are more negative (-1.4 to -1.0 V) than what is typically expected of [4Fe-4S] ferredoxin proteins. Instead, we find that ?(2)-bound states are lower in energy than ROH-bound states when the [4Fe-4S] cluster is 1e(-) reduced. Furthermore, ?(2)-bound states can already be generated in the oxidized state, yielding reduction potentials of ca. -700 mV when electron addition occurs after rotation of the HMBPP C4-OH group. We demonstrate that such ?(2)-bound states are kinetically accessible both when the IspH [4Fe-4S] cluster is oxidized and 1e(-) reduced. The energetically preferred pathway gives 1e(-) reduction of the cluster after substrate conformational change, generating the 1e(-) reduced intermediate proposed in the organometallic mechanism.

Dataset Information

Mechanistic insights into the reductive dehydroxylation pathway for the biosynthesis of isoprenoids promoted by the IspH enzyme.

Publications

Mechanistic insights into the reductive dehydroxylation pathway for the biosynthesis of isoprenoids promoted by the IspH enzyme.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets