Myriam Seemann

Isoprenoid biosynthesis via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB/IspH) from Escherichia coli is a [4Fe–4S] protein

The last enzyme (LytB) of the methylerythritol phosphate pathway for isoprenoid biosynthesis catalyzes the reduction of (E)‐4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate into isopentenyl diphosphate and dimethylallyl diphosphate. This enzyme possesses a dioxygen‐sensitive [4Fe–4S] cluster. This prosthetic group was characterized in the Escherichia coli enzyme by UV/visible and electron paramagnetic resonance spectroscopy after reconstitution of the purified protein. Enzymatic activity required the presence of a reducing system such as flavodoxin/flavodoxin reductase/reduced nicotinamide adenine dinucleotide phosphate or the photoreduced deazaflavin radical.

Isoprenoid Biosynthesis through the Methylerythritol Phosphate Pathway: The (E)‐4‐Hydroxy‐3‐methylbut‐2‐enyl Diphosphate Synthase (GcpE) is a [4Fe–4S] Protein

nitrilotriaceticacidagarosecolumn. The enzyme was found to be 95% pure by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electro-phoresis) and presented an apparent molecular mass of43kDa. The purified protein was inactive, even in thepresenceofthereducingsystemsdetailedbelow.Suchalackofcatalyticactivitywasprobablyaresultofthepredominant

Isoprenoid biosynthesis in plant chloroplasts via the MEP pathway: Direct thylakoid/ferredoxin-dependent photoreduction of GcpE/IspG

In the methylerythritol phosphate pathway for isoprenoid biosynthesis, the GcpE/IspG enzyme catalyzes the conversion of 2‐C‐methyl‐d‐erythritol 2,4‐cyclodiphosphate into (E)‐4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate. This reaction requires a double one‐electron transfer involving a [4Fe–4S] cluster. A thylakoid preparation from spinach chloroplasts was capable in the presence of light to act as sole electron donor for the plant GcpE Arabidopsis thaliana in the absence of any pyridine nucleotide. This is in sharp contrast with the bacterial Escherichia coli GcpE, which requires flavodoxin/flavodoxin reductase and NADPH as reducing system and represents the first proof that the electron flow from photosynthesis can directly act in phototrophic organisms as reducer in the 2‐C‐methyl‐d‐erythritol 4‐phosphate pathway, most probably via ferredoxin, in the absence of any reducing cofactor. In the dark, the plant GcpE catalysis requires in addition of ferredoxin NADP+/ferredoxin oxido‐reductase and NADPH as electron shuttle.

Identification of gcpE as a novel gene of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis in Escherichia coli.

The 2‐C‐methyl‐D‐erythritol 4‐phosphate (MEP) pathway for isoprenoid biosynthesis is essential in most eubacteria and plants and has remarkable biotechnological interest. However, only the first steps of this pathway have been determined. Using bioinformatic and genetic approaches, we have identified gcpE as a novel gene of the MEP pathway. The distribution of this gene in bacteria and plants strictly parallels that of the gene encoding 1‐deoxy‐D‐xylulose 5‐phosphate reductoisomerase, which catalyses the first committed step of the MEP pathway. Our data demonstrate that the gcpE gene is essential for the MEP pathway in Escherichia coli and indicate that this gene is required for the trunk line of the isoprenoid biosynthetic route.

Isoprenoid biosynthesis via the methylerythritol phosphate pathway. (E)-4-Hydroxy-3-methylbut-2-enyl diphosphate: chemical synthesis and formation from methylerythritol cyclodiphosphate by a cell-free system from Escherichia coli

Abstract 2- C -Methyl- d -erythritol cyclodiphosphate is converted into ( E )-4-hydroxy-3-methylbut-2-enyl diphosphate by a cell-free system from an Escherichia coli strain overexpressing the gcpE gene. The latter diphosphate, representing probably the last intermediate in the MEP pathway for isoprenoid biosynthesis, was identified by comparison with reference material obtained by chemical synthesis.

Isoprenoid biosynthesis in chloroplasts via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE) from Arabidopsis thaliana is a [4Fe–4S] protein

The mevalonate-independent methylerythritol phosphate pathway is widespread in bacteria. It is also present in the chloroplasts of all phototrophic organisms. Whereas the first steps, are rather well known, GcpE and LytB, the enzymes catalyzing the last two steps have been much less investigated. 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate is transformed by GcpE into 4-hydroxy-3-methylbut-2-enyl diphosphate, which is converted by LytB into isopentenyl diphosphate or dimethylallyl diphosphate. Only the bacterial GcpE and LytB enzymes have been investigated to some extent, but nothing is known about the corresponding plant enzymes. In this contribution, the prosthetic group of GcpE from the plant Arabidopsis thaliana and the bacterium Escherichia coli has been fully characterized by Mössbauer spectroscopy after reconstitution with 57FeCl3, Na2S and dithiothreitol. It corresponds to a [4Fe-4S] cluster, suggesting that both plant and bacterial enzymes catalyze the reduction of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate via two consecutive one-electron transfers. In contrast to the bacterial enzyme, which utilizes NADPH/flavodoxin/flavodoxin reductase as a reducing shuttle system, the plant enzyme could not use this reduction system. Enzymatic activity was only detected in the presence of the 5-deazaflavin semiquinone radical.

Isoprenoid Biosynthesis via the MEP Pathway: In Vivo Mössbauer Spectroscopy Identifies a [4Fe-4S]2+ Center with Unusual Coordination Sphere in the LytB Protein

The MEP pathway for the biosynthesis of isoprene units is present in most pathogenic bacteria, in the parasite responsible for malaria, and in plant plastids. This pathway is absent in animals and is accordingly a target for the development of antimicrobial drugs. LytB, also called IspH, the last enzyme of this pathway catalyzes the conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) using an oxygen sensitive iron sulfur cluster. The exact nature of this iron sulfur cluster is still a matter of debate. We have used (57)Fe Mössbauer spectroscopy to investigate the LytB cluster in whole E. coli cells and in the anaerobically purified enzyme: In LytB an unusual [4Fe-4S](2+) cluster is attached to the protein by three conserved cysteines and contains a hexacoordinated iron linked to three sulfurs of the cluster and three additional oxygen or nitrogen ligands.

Accumulation of 2‐C‐methyl‐d‐erythritol 2,4‐cyclodiphosphate in illuminated plant leaves at supraoptimal temperatures reveals a bottleneck of the prokaryotic methylerythritol 4‐phosphate pathway of isoprenoid biosynthesis

Metabolic profiling using phosphorus nuclear magnetic resonance ((31)P-NMR) revealed that the leaves of different herbs and trees accumulate 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcDP), an intermediate of the methylerythritol 4-phosphate (MEP) pathway, during bright and hot days. In spinach (Spinacia oleracea L.) leaves, its accumulation closely depended on irradiance and temperature. MEcDP was the only (31)P-NMR-detected MEP pathway intermediate. It remained in chloroplasts and was a sink for phosphate. The accumulation of MEcDP suggested that its conversion rate into 4-hydroxy-3-methylbut-2-enyl diphosphate, catalysed by (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE), was limiting under oxidative stress. Indeed, O(2) and ROS produced by photosynthesis damage this O(2)-hypersensitive [4Fe-4S]-protein. Nevertheless, as isoprenoid synthesis was not inhibited, damages were supposed to be continuously repaired. On the contrary, in the presence of cadmium that reinforced MEcDP accumulation, the MEP pathway was blocked. In vitro studies showed that Cd(2+) does not react directly with fully assembled GcpE, but interferes with its reconstitution from recombinant GcpE apoprotein and prosthetic group. Our results suggest that MEcDP accumulation in leaves may originate from both GcpE sensitivity to oxidative environment and limitations of its repair. We propose a model wherein GcpE turnover represents a bottleneck of the MEP pathway in plant leaves simultaneously exposed to high irradiance and hot temperature.

Isoprenoid biosynthesis in Escherichia coli via the methylerythritol phosphate pathway: enzymatic conversion of methylerythritol cyclodiphosphate into a phosphorylated derivative of (E)-2-methylbut-2-ene-1,4-diol

Abstract A crude cell-free system from an Escherichia coli strain overexpressing the cluster containing the three genes yfgA, yfgB, and gcpE converted 2-C-methyl- d -erythritol 2,4-cyclodiphosphate ( 1 ) into a phosphorylated derivative of (E)-2-methylbut-2-ene-1,4-diol ( 6 ), which most probably represents a novel intermediate in the methylerythritol phosphate pathway for isoprenoid biosynthesis. The free diol 6 was accumulated by phosphatase treatment of the crude enzyme preparation and was identified by comparison with a synthetic reference.

Isoprenoid biosynthesis via the methylerythritol phosphate pathway: accumulation of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate in a gcpE deficient mutant of Escherichia coli

Abstract In the bacterium Escherichia coli , gcpE is an essential gene in the methylerythritol phosphate pathway for isoprenoid biosynthesis. Incubation of [1- 3 H]methylerythritol with an E . coli mutant defective in the gcpE gene resulted in the accumulation of [1- 3 H]methylerythritol 2,4-cyclodiphosphate. This suggests that the GCPE protein is involved in the further conversion of methylerythritol cyclodiphosphate into isoprenoids.