LytB protein catalyzes the terminal step of the 2‐<i>C</i>‐methyl‐<scp>D</scp>‐erythritol‐4‐phosphate pathway of isoprenoid biosynthesis

210

234

Earlier in vivo studies have shown that the sequential action of the IspG and IspH proteins is essential for the reductive transformation of 2C-methyl-D-erythritol 2,4-cyclodiphosphate into dimethylallyl diphosphate and isopentenyl diphosphate via 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. A recombinant fusion protein comprising maltose binding protein and IspG protein domains was purified from a recombinant Escherichia coli strain. The purified protein failed to transform 2C-methyl-D-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate, but catalytic activity could be restored by the addition of crude cell extract from an ispG-deficient E. coli mutant. This indicates that auxiliary proteins are required, probably as shuttles for redox equivalents. On activation by photoreduced 10-methyl-5-deazaisoalloxazine, the recombinant protein catalyzed the formation of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate from 2C-methyl-D-erythritol 2,4-cyclodiphosphate at a rate of 1 nmol⅐min ؊1 ⅐mg ؊1 . Similarly, activation by photoreduced 10-methyl-5-deaza-isoalloxazine enabled purified IspH protein to catalyze the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into a 6:1 mixture of isopentenyl diphosphate and dimethylallyl diphosphate at a rate of 0.4 mol⅐min ؊1 ⅐mg ؊1 . IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of 3 nmol⅐min ؊1 ⅐mg ؊1 . The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions.

Section: Discussionsupporting

confidence: 83%

The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: Studies on the mechanisms of the reactions catalyzed by IspG and IspH protein

Rohdich

Zepeck

Adam

et al. 2003

210

234

“…In the more recently discovered nonmevalonate pathway, the terminal intermediate, 1-hydroxy-2-methyl-2-butenyl 4-diphosphate (HMBPPFig. 1), is reductively converted into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by the action of a single, monomeric iron-sulfur protein specified by the ispH gene (initially designated lytB) (3,4). This pathway is crucial for most pathogenic bacteria as well as for Plasmodium falciparum (the parasite causing malaria) and plant plastids, yet it is absent in animals, and accordingly IspH has received considerable attention as potential drug target.…”

mentioning

confidence: 99%

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

Gräwert

Span

Eisenreich

et al. 2009

102

228

Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) represent the two central intermediates in the biosynthesis of isoprenoids. The recently discovered deoxyxylulose 5-phosphate pathway generates a mixture of IPP and DMAPP in its final step by reductive dehydroxylation of 1-hydroxy-2-methyl-2-butenyl 4-diphosphate. This conversion is catalyzed by IspH protein comprising a central iron-sulfur cluster as electron transfer cofactor in the active site. The five crystal structures of IspH in complex with substrate, converted substrate, products and PP i reported in this article provide unique insights into the mechanism of this enzyme. While IspH protein crystallizes with substrate bound to a [4Fe-4S] cluster, crystals of IspH in complex with IPP, DMAPP or inorganic pyrophosphate feature [3Fe-4S] clusters. The IspH:substrate complex reveals a hairpin conformation of the ligand with the C(1) hydroxyl group coordinated to the unique site in a [4Fe-4S] cluster of aconitase type. The resulting alkoxide complex is coupled to a hydrogen-bonding network, which serves as proton reservoir via a Thr167 proton relay. Prolonged x-ray irradiation leads to cleavage of the C(1)-O bond (initiated by reducing photo electrons). The data suggest a reaction mechanism involving a combination of Lewis-acid activation and proton coupled electron transfer. The resulting allyl radical intermediate can acquire a second electron via the iron-sulfur cluster. The reaction may be terminated by the transfer of a proton from the β-phosphate of the substrate to C(1) (affording DMAPP) or C(3) (affording IPP).iron-sulfur protein | LytB protein | nonmevalonate pathway | terpene biosynthesis | isoprenoid biosynthesis

“…Next, CDP-ME2P is converted to 2C-methyl-D-erythritol-2,4-cyclodiphosphate and CMP (18,19). The cyclodiphosphate then, in two enzyme-catalyzed stages, undergoes reduction and elimination to 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate and onto IPP and DMAPP (20)(21)(22)(23).…”

mentioning

confidence: 99%

Biosynthesis of isoprenoids: Crystal structure of 4-diphosphocytidyl-2C-methyl- d -erythritol kinase

Miallau

Alphey

Kemp

et al. 2003

109

4-Diphosphocytidyl-2C-methyl-D-erythritol kinase, an essential enzyme in the nonmevalonate pathway of isopentenyl diphosphate and dimethylallyl diphosphate biosynthesis, catalyzes the single ATP-dependent phosphorylation stage affording 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate. The 2-Å resolution crystal structure of the Escherichia coli enzyme in a ternary complex with substrate and a nonhydrolyzable ATP analogue reveals the molecular determinants of specificity and catalysis. The enzyme subunit displays the ␣͞␤ fold characteristic of the galactose kinase͞ homoserine kinase͞mevalonate kinase͞phosphomevalonate kinase superfamily, arranged into cofactor and substrate-binding domains with the catalytic center positioned in a deep cleft between domains. Comparisons with related members of this superfamily indicate that the core regions of each domain are conserved, whereas there are significant differences in the substrate-binding pockets. The nonmevalonate pathway is essential in many microbial pathogens and distinct from the mevalonate pathway used by mammals. The high degree of sequence conservation of the enzyme across bacterial species suggests similarities in structure, specificity, and mechanism. Our model therefore provides an accurate template to facilitate the structure-based design of broad-spectrum antimicrobial agents.galactose kinase͞homoserine kinase͞mevalonate kinase͞ phosphomevalonate kinase ͉ enzyme mechanism ͉ nonmevalonate ͉ phosphorylation T he AT P-dependent 4-diphosphocytidyl-2C-methyl-Derythritol (CDP-ME) kinase (EC 2.7.1.148) participates in the biosynthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). These isomers are the universal five-carbon precursors of isoprenoids, a diverse and important family of natural products that includes sterols, dolichols, triterpenes, and ubiquinones, and components of macromolecules such as the prenyl groups of prenylated proteins and isopentenylated tRNAs (1-3). Isoprenoids contribute to many biological functions, including electron transport in respiration and photosynthesis, hormone-based signaling, apoptosis, meiosis, protein cleavage, and degradation (4). In addition, they provide important structural components of cell membranes (1).Two biosynthetic routes to IPP and DMAPP have evolved. In eukaryotes, archaebacteria, and a few eubacteria, the precursor biosynthesis is through the mevalonate pathway (3-7). This begins with the conversion of three molecules of acetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA followed by reduction, phosphorylation, and decarboxylation to generate IPP, some of which is isomerized to DMAPP. The last three steps in this pathway are ATP-dependent and catalyzed by the structurally related mevalonate kinase (MVK), phosphomevalonate kinase, and mevalonate 5-diphosphate decarboxylase.In chloroplasts, algae, cyanobacteria, most eubacteria, and the apicomplexa, IPP and DMAPP synthesis is accomplished by seven enzymes in a pathway named after one of the intermediates, the 1-deoxy-D-xylulose-5-ph...