The non-mevalonate or also called MEP pathway is an essential route for the biosynthesis of isoprenoid precursors in most bacteria and in microorganisms belonging to the Apicomplexa phylum, such as ...the parasite responsible for malaria. The absence of this pathway in mammalians makes it an interesting target for the discovery of novel anti-infectives. As last enzyme of this pathway, IspH is an oxygen sensitive 4Fe-4S metalloenzyme that catalyzes 2H
/2e
reductions and a water elimination by involving non-conventional bioinorganic and bioorganometallic intermediates. After a detailed description of the discovery of the 4Fe-4S cluster of IspH, this review focuses on the IspH mechanism discussing the results that have been obtained in the last decades using an approach combining chemistry, enzymology, crystallography, spectroscopies, and docking calculations. Considering the interesting druggability of this enzyme, a section about the inhibitors of IspH discovered up to now is reported as well. The presented results constitute a useful and rational help to inaugurate the design and development of new potential chemotherapeutics against pathogenic organisms.
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.
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.
Incorporation of 13C-labeled glycerol or pyruvate into the ubiquinone Q8 of Escherichia coli mutants lacking enzymes of the triose phosphate metabolism and of (U-13C6)glucose into the triterpenoids ...of the hopane series of Zymomonas mobilis showed that glyceraldehyde 3-phosphate (or eventually glyceraldehyde) and a C2 unit derived from pyruvate decarboxylation were the only precursors of the C5 skeleton of isoprenic units in a novel non-mevalonate pathway for isoprenoid biosynthesis in these bacteria.
The ethanol-producing bacterium Zymomonas mobilis has attracted considerable scientific and commercial interest due to its exceptional physiological properties. Shuttle vectors derived from native ...plasmids have previously been successfully used for heterologous gene expression in this bacterium for a variety of purposes, most notably for metabolic engineering applications.
A quantitative PCR (qPCR) approach was used to determine the copy numbers of two endogenous double stranded DNA plasmids: pZMO1A (1,647 bp) and pZMO7 (pZA1003; 4,551 bp) within the NCIMB 11163 strain of Z. mobilis. Data indicated pZMO1A and pZMO7 were present at ca. 3-5 and ca. 1-2 copies per cell, respectively. A ca. 1,900 bp fragment from plasmid pZMO7 was used to construct two Escherichia coli - Z. mobilis shuttle vectors (pZ7C and pZ7-184). The intracellular stabilities and copy numbers of pZ7C and pZ7-184 were characterized within the NCIMB 11163, ATCC 29191 and (ATCC 10988-derived) CU1 Rif2 strains of Z. mobilis. Both shuttle vectors could be stably maintained within the ATCC 29191 strain (ca. 20-40 copies per cell), and the CU1 Rif2 strain (ca. 2-3 copies per cell), for more than 50 generations in the absence of an antibiotic selectable marker. A selectable marker was required for shuttle vector maintenance in the parental NCIMB 11163 strain; most probably due to competition for replication with the endogenous pZMO7 plasmid molecules. N-terminal glutathione S-transferase (GST)-fusions of four endogenous proteins, namely the acyl-carrier protein (AcpP); 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA); DNA polymerase III chi subunit (HolC); and the RNA chaperone protein Hfq; were successfully expressed from pZ7C-derived shuttle vectors, and their protein-protein binding interactions were analyzed in Z. mobilis ATCC 29191. Using this approach, proteins that co-purified with AcpP and KdsA were identified.
We show that a shuttle vector-based protein affinity 'pull-down' approach can be used to probe protein interaction networks in Z. mobilis cells. Our results demonstrate that protein expression plasmids derived from pZMO7 have significant potential for use in future biological or biotechnological applications within Z. mobilis.
One of the last bottlenecks in the elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis has been solved. A 4Fe–4S cluster is the prosthetic group of the GcpE protein from ...E. coli. This enzyme is involved in the conversion of 1 into 2, through two successive one‐electron transfers.
Isoprenoid biosynthesis was investigated in the green alga Scenedesmus obliques grown heterotrophically on 13C-labelled glucose and acetate. Several isoprenoid compounds were isolated and ...investigated by 13C-NMR spectroscopy. According to the 13C-labelling pattern indicated by the 13C-NMR spectra, the biosynthesis of all plastidic isoprenoids investigated (prenyl sidechains of chlorophylls and plastoquinone-9, and the carotenoids beta-carotene and lutein), as well as of the non-plastidic cytoplasmic sterols, does not proceed via the classical acetate/mevalonate pathway (which leads from acetyl-CoA via mevalonate to isopentenyl diphosphate), but via the novel glyceraldehyde 3-phosphate/pyruvate route recently detected in eubacteria. Formation of isopentenyl diphosphate involves the condensation of a C2 unit derived from pyruvate decarboxylation with glyceraldehyde 3-phosphate and a transposition yielding the branched C5 skeleton of isoprenic units.
Bacteria have evolved several outstanding strategies to resist to compounds or factors that compromise their survival. The first line of defense of the cell against environmental stresses is the ...membrane with fatty acids as fundamental building blocks of phospholipids. In this review, we focus on a periplasmic heme enzyme that catalyzes the cis-trans isomerization of unsaturated fatty acids to trigger a decrease in the fluidity of the membrane in order to rapidly counteract the danger. We particularly detailed the occurrence of such cis-trans isomerase in Nature, the different stresses that are at the origin of the double bond isomerization, the first steps in the elucidation of the mechanism of this peculiar metalloenzyme and some aspects of its regulation.
The graphical abstract describes the action of the cis-trans isomerase of fatty acids that allows some bacteria to shield themselves from many stresses by triggering a sudden rigidification of their membrane due to the trans geometry of the fatty acid double bond. Display omitted
•Production of trans-unsaturated fatty acids from cis-unsaturated fatty acids•A fast adaptive response of some bacteria to resist to environmental stresses•Bacterial survival by decreasing membrane fluidity to rapidly respond to the danger•The cis-trans isomerase of unsaturated fatty acids is a cytochrome-c type enzyme.
For the ammonia-oxidizing bacterium
Nitrosomonas europaea, grown autotrophically using semicontinuous culturing, average biomass was depleted in
13C relative to CO
2 dissolved in the medium by ca. ...20‰ and the total-lipid extract was depleted in
13C relative to biomass by 3.7‰. The
n-alkyl lipids (weighted average of fatty acids) and isoprenoid lipids (weighted average of hopanoids) were both depleted in
13C relative to biomass by about 9‰. The large depletion in the isoprenoid lipids seems to indicate that isotopic fractionations associated with the biosynthesis of methylerythritol phosphate (MEP) affected at least two carbon positions in each isoprene unit. Among the fatty acids,
trans-9-hexadecenoic acid was most depleted (13.0‰ relative to biomass), followed by
cis-9- hexadecenoic acid (9.6‰) and hexadecanoic acid (6.9‰). Isotopic relationships between the three acids suggest that significant isotope effects were associated with the desaturation and
cis to
trans isomerization of fatty acids. Given these observations, hopanoids produced by ammonia-oxidizing bacteria growing in natural waters are likely to be depleted in
13C by 26–30‰ relative to dissolved CO
2. Since CO
2 at aquatic oxyclines is often depleted in
13C, the range of
δ values expected for hopanoids is ca. −34‰ to −55‰. The
δ values of geohopanoids observed in numerous studies and attributed to unspecified chemoautotrophs fall within this range.
The non-mevalonate or also called MEP pathway is an essential route for the biosynthesis of isoprenoid precursors in most bacteria and in microorganisms belonging to the Apicomplexa phylum, such as ...the parasite responsible for malaria. The absence of this pathway in mammalians makes it an interesting target for the discovery of novel anti-infectives. As last enzyme of this pathway, IspH is an oxygen sensitive 4Fe-4S metalloenzyme that catalyzes 2H+/2e- reductions and a water elimination by involving non-conventional bioinorganic and bioorganometallic intermediates. After a detailed description of the discovery of the 4Fe-4S cluster of IspH, this review focuses on the IspH mechanism discussing the results that have been obtained in the last decades using an approach combining chemistry, enzymology, crystallography, spectroscopies, and docking calculations. Considering the interesting druggability of this enzyme, a section about the inhibitors of IspH discovered up to now is reported as well. The presented results constitute a useful and rational help to inaugurate the design and development of new potential chemotherapeutics against pathogenic organisms.
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