Functional characterization of GcpE, an essential enzyme of the non‐mevalonate pathway of isoprenoid biosynthesis

Kollas, Ann-Kristin; Duin, Evert C.; Eberl, Matthias; Altincicek, Boran; Hintz, Martin; Reichenberg, Armin; Henschker, Dajana; Henne, Anke; Steinbrecher, Irina; Ostrovsky, D. N.; Hedderich, Reiner; Beck, Ewald; Jomaa, Hassan; Wiesner, Jochen

doi:10.1016/s0014-5793(02)03725-0

Cited by 103 publications

(109 citation statements)

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“…Based on the similarity of the EPR signals in GcpE and FTR we propose that the transient signal detected in GcpE is due to binding and stabilization of a radical-type intermediate by the 4Fe cluster. This is an important finding since several mechanisms have been proposed in the literature [10,13,16,33], but in only two papers was a direct role proposed for the 4Fe cluster [11,34].…”

Section: Resultsmentioning

confidence: 91%

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Possible direct involvement of the active‐site [4Fe–4S] cluster of the GcpE enzyme from Thermus thermophilus in the conversion of MEcPP

Adedeji¹,

Hernandez²,

Wiesner³

et al. 2006

FEBS Letters

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The GcpE enzyme converts 2-C-methyl-D D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) in the penultimate step of the DOXP pathway for isoprene biosynthesis. Purification of the enzyme under exclusion of air leads to a preparation that contains solely [4Fe-4S] clusters. Kinetic studies showed that in the presence of the artificial reductant dithionite and MEcPP a new transient iron-sulfur-based signal is detected in electron paramagnetic resonance (EPR) spectroscopy. Similarity of this EPR signal to that detected in ferredoxin:thioredoxin reductase indicates that during the reaction an intermediate is directly bound to the active-site cluster.

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Section: Resultsmentioning

confidence: 91%

“…The gcpE gene of T. thermophilus was overexpressed in an E. coli host and the gcpE product was purified as described previously [10]. This method generally yielded 25 mg of GcpE in 10 ml.…”

Section: Methodsmentioning

confidence: 99%

Possible direct involvement of the active‐site [4Fe–4S] cluster of the GcpE enzyme from Thermus thermophilus in the conversion of MEcPP

Adedeji¹,

Hernandez²,

Wiesner³

et al. 2006

FEBS Letters

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“…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

Proc. Natl. Acad. Sci. U.S.A.

109

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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...

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“…Recent data has shown that (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE) and LytB proteins are iron-sulfur proteins containing a [4Fe-4S] 2ϩ cluster after reconstitution of the purified protein (12)(13)(14). GcpE catalyzes the reduction of MEcPP into HMBPP via two successive one-electron transfers (11,13). The last step of the MEP pathway is catalyzed by IspH (or LytB), which converts HMBPP into isopentenyl diphosphate or dimethylallyl diphosphate via two successive one-electron transfers (15).…”

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confidence: 99%

Cyanobacterial Non-mevalonate Pathway

Okada

Hase

2005

Journal of Biological Chemistry

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(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE), which catalyzes the conversion of 2-Cmethyl-D-erythritol cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP), is an essential enzyme of the non-mevalonate (2-C-methyl-D-erythritol-4-phosphate (MEP)) pathway for isoprenoid biosynthesis. The terminal steps of the MEP pathway are still not fully understood, although this pathway is necessary for survival in various organisms such as cyanobacteria, plastids of algae and higher plants, and the apicoplast of human malaria parasites. To determine the efficient redox partner for thermophilic cyanobacterial GcpE, We have expressed the gcpE and petF genes in Escherichia coli and studied the protein-protein interaction of GcpE protein with ferredoxin I (PetF) from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. Recombinant GcpE protein was purified by an N-terminal His 6 tag and reconstituted as a [4Fe-4S] 2؉ metalloprotein. GcpE was shown to interact strongly with PetF via the bacterial two-hybrid system designed to detect protein-protein interactions. Moreover, a direct protein-protein interaction between PetF and GcpE was confirmed in an in vitro glutathione Stransferase (GST) pull-down assay. To investigate electron transfer activity from PetF to GcpE, we also constructed a NADPH-dependent reducing shuttle system with purified recombinant ferredoxin-NADP ؉ oxidoreductase (PetH) and PetF. The result demonstrated that PetF has the ability to transfer electrons to GcpE. Thus, the combined data provide the first evidence that GcpE is a ferredoxin-dependent enzyme in T. elongatus BP-1.

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Functional characterization of GcpE, an essential enzyme of the non‐mevalonate pathway of isoprenoid biosynthesis

Cited by 103 publications

References 20 publications

Possible direct involvement of the active‐site [4Fe–4S] cluster of the GcpE enzyme from Thermus thermophilus in the conversion of MEcPP

Possible direct involvement of the active‐site [4Fe–4S] cluster of the GcpE enzyme from Thermus thermophilus in the conversion of MEcPP

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

Cyanobacterial Non-mevalonate Pathway

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