Michel Rohmer

The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants†

1 Isoprenoid biosynthesis 1.1 The mevalonate route to isopentenyl diphosphate 1.2 Isoprenoid biosynthesis in higher plants: some contradictions with the mevalonate pathway 2 The discovery of the mevalonate-independent pathway 2.1 The origin of the discovery: the biosynthesis of bacterial hopanoids 2.2 The origin of the carbon atoms of isoprenic units in the mevalonate-independent pathway 2.3 d-Glyceraldehyde 3-phosphate and pyruvate as the first precursors of isopentenyl diphosphate 3 Towards the identification of intermediates and enzymes of the new pathway 3.1 1-Deoxy-d-xylulose 5-phosphate and 1-deoxy-d-xylulose 5-phosphate synthase 3.2 2-C-Methyl-d-erythritol 4-phosphate and 1-deoxy-d-xylulose 5-phosphate reducto-isomerase 4 The distribution of the glyceraldehyde 3-phosphate/pyruvate pathway amongst prokaryotes 5 The distribution of the GAP/pyruvate pathway amongst phototrophic eukaryotes 5.1 Essential plant chloroplast isoprenoids and sterols from green algae 5.2 Isoprenoids from secondary metabolism 5.3 Intermediate exchanges between the mevalonate and the GAP/pyruvate pathways in plants 6 Conclusion 7 Acknowledgments 8 References

In vitro antimicrobial activity of extracts and compounds of some selected medicinal plants from Cameroon

AIM OF THE STUDY Seven extracts and eight compounds from four selected Cameroonian medicinal plants, Solanecio mannii Hook f. (Asteraceae), Monodora myristica Dunal (Annonaceae), Albizia gummifera (J.F. Gmel) C.A. Smith (Fabaceae/Mimosoideae) and Glyphaea brevis (Spreng) Monachino (Tiliaceae), traditionally used for the treatment of hepatitis, parasites and other infectious diseases, were tested in vitro for their antimicrobial activity against Gram-positive (5 species) and Gram-negative (4 species) bacteria species and pathogenic yeasts (2 Candida species), to establish whether or not they have antimicrobial activity and to validate scientifically their use in traditional medicine. MATERIALS AND METHODS The agar disc diffusion and the microbroth dilution methods were used to determine the zone of inhibition between the edge of the filter paper and the edge of the inhibition area (IZ) and the minimal inhibitory concentration (MIC) respectively. RESULTS The most active extracts against Candida albicans and Candida krusei were respectively the cyclohexane extract from the fruits of Monodora myristica and the ethyl acetate extract from the stem bark of Albizia gummifera (MIC=6.3 microg/ml for both extracts). The lowest MIC value (1.6 microg/ml) for purified compounds was obtained on Candida albicans with a mixture of linear aliphatic primary alcohols (n-C24H50O to n-C30H62O), with n-hexacosanol (1b) as major compound and mixture of fatty acid esters of diunsaturated linear 1,2-diols (6). CONCLUSION These results afford ground informations for the potential use of the crude extracts of these species as well as of some of the isolated compounds in bacterial and fungal infections.

Plant isoprenoid biosynthesis via the MEP pathway: In vivo IPP/DMAPP ratio produced by (E)‐4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate reductase in tobacco BY‐2 cell cultures

Feeding tobacco BY‐2 cells with [2‐13C,4‐2H]deoxyxylulose revealed from the 13C labeling that the plastid isoprenoids, synthesized via the MEP pathway, are essentially derived from the labeled precursor. The ca. 15% 2H retention observed in all isoprene units corresponds to the isopentenyl diphosphate (IPP)/dimethylallyl diphosphate (DMAPP) ratio (85:15) directly produced by the hydroxymethylbutenyl diphosphate reductase, the last enzyme of the MEP pathway. 2H retention characterizes the isoprene units derived from the DMAPP branch, whereas 2H loss represents the signature of the IPP branch. Taking into account the enantioselectivity of the reactions catalyzed by the (E)‐4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate reductase, the IPP isomerase and the trans‐prenyl transferase, a single biogenetic scheme allows to interpret all labeling patterns observed in bacteria or plants upon incubation with 2H labeled deoxyxylulose.

Biosynthesis of monoterpene scent compounds in roses

Stop to smell the roses Some roses smell beautiful, yet others only look beautiful. Magnard et al. leveraged this distinction to study the biosynthesis of geraniol, a monoterpene alcohol in rose scent (see the Perspective by Tholl and Gershenzon). Enzymes known for geraniol synthesis in other plants, such as basil, did not seem to provide that function for roses. Instead, a diphosphohydrolase, which functions in the cytoplasm of cells in rose petals, generates the geraniol emitted by fragrant roses. Identification of the enzyme and its gene enables marker-assisted breeding to put the perfume back into beauty. Science, this issue p. 81; see also p. 28 Roses that are showy may not be fragrant if this enzyme is missing. [Also see Perspective by Tholl and Gershenzon] The scent of roses (Rosa x hybrida) is composed of hundreds of volatile molecules. Monoterpenes represent up to 70% percent of the scent content in some cultivars, such as the Papa Meilland rose. Monoterpene biosynthesis in plants relies on plastid-localized terpene synthases. Combining transcriptomic and genetic approaches, we show that the Nudix hydrolase RhNUDX1, localized in the cytoplasm, is part of a pathway for the biosynthesis of free monoterpene alcohols that contribute to fragrance in roses. The RhNUDX1 protein shows geranyl diphosphate diphosphohydrolase activity in vitro and supports geraniol biosynthesis in planta.

Biosynthesis of isoprene units: Mössbauer spectroscopy of substrate and inhibitor binding to the [4Fe-4S] cluster of the LytB/IspH enzyme.

The biosynthesis of isoprenoids in many bacteria and in the malaria parasite Plasmodium falciparum occurs according to the methylerythritol phosphate (MEP) pathway,[1] an alternative to the mevalonate pathway.[2] The MEP pathway is a valuable target for the development of new antimicrobial agents as it is essential for microorganisms, and absent in humans.[3] In the last step of this biosynthetic route, HMBPP 1 is converted into a mixture of IPP and DMAPP, which are both precursors of isoprenoids (Scheme 1). This reaction is catalyzed by a peculiar [4Fe-4S] center of the LytB/IspH protein. [4]

Inhibition of IspH, a [4Fe–4S]2+ Enzyme Involved in the Biosynthesis of Isoprenoids via the Methylerythritol Phosphate Pathway

The MEP pathway, which is absent in animals but present in most pathogenic bacteria, in the parasite responsible for malaria and in plant plastids, is a target for the development of antimicrobial drugs. IspH, an oxygen-sensitive [4Fe-4S] enzyme, catalyzes the last step of this pathway and converts (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A crucial step in the mechanism of this enzyme is the binding of the C4 hydroxyl of HMBPP to the unique fourth iron site in the [4Fe-4S](2+) moiety. Here, we report the synthesis and the kinetic investigations of two new extremely potent inhibitors of E. coli IspH where the OH group of HMBPP is replaced by an amino and a thiol group. (E)-4-Mercapto-3-methylbut-2-en-1-yl diphosphate is a reversible tight-binding inhibitor of IspH with K(i) = 20 ± 2 nM. A detailed kinetic analysis revealed that (E)-4-amino-3-methylbut-2-en-1-yl diphosphate is a reversible slow-binding inhibitor of IspH with K(i) = 54 ± 19 nM. The slow binding behavior of this inhibitor is best described by a one-step mechanism with the slow step consisting of the formation of the enzyme-inhibitor (EI) complex.

The mevalonate-independent methylerythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis, including carotenoids

A mevalonate-independent route to isopentenyl diphosphate (IPP), the universal precursor of isoprenoids, is present in many bacteria, in some unicellular green algae and in the plant plastids. All essential isoprenoids related to photosynthesis, including the carotenoids, are synthesized via this alternative metabolic route. IPP is formed from pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate and 2-C-methyl-D- erythritol 4-phosphate. Later steps are still poorly known, although extensive data on the origin of the hydrogen atoms of IPP are now available.

Measurement of carbon flux through the MEP pathway for isoprenoid synthesis by 31P‐NMR spectroscopy after specific inhibition of 2‐C‐methyl‐d‐erythritol 2,4‐cyclodiphosphate reductase. Effect of light and temperature

The methylerythritol 4-phosphate (MEP) and the mevalonate pathways are the unique synthesis routes for the precursors of all isoprenoids. An original mean to measure the carbon flux through the MEP pathway in plants is proposed by using cadmium as a total short-term inhibitor of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) reductase (GcpE) and measuring the accumulation rate of its substrate MEcDP by (31) P-NMR spectroscopy. The MEP pathway metabolic flux was determined in spinach (Spinacia oleracea), pea (Pisum sativum), Oregon grape (Mahonia aquifolium) and boxwood (Buxus sempervirens) leaves. In spinach, flux values were compared with the synthesis rate of major isoprenoids. The flux increases with light intensity (fourfold in the 200-1200 µmol m(-2) s(-1) PPFR range) and temperature (sevenfold in the 25-37 °C range). The relationship with the light and the temperature dependency of isoprenoid production downstream of the MEP pathway is discussed.

Methylerythritol Phosphate Pathway

Isoprenoids represent the most diverse natural products family. Their carbon skeleton is formally derived from the branched C5 isoprene skeleton. Two metabolic pathways are utilized by living organisms for synthesizing isopentenyl diphosphate (IPP) and dimethylallyl diphosphate, the universal precursors of this series. In animals, fungi, a few bacteria, and in the cytoplasm of the phototrophic organisms, acetyl coenzyme A and mevalonate (MVA), as the committed intermediate, are the precursors of isoprene units. In most bacteria and in the plastids of phototrophic organisms and related phyla, isoprene units are derived from carbohydrate metabolism. Glyceraldehyde phosphate and pyruvate serve as starting material, leading to methylerythritol phosphate (MEP), the key intermediate of this alternative pathway. In the first issue of this encyclopedia, two chapters were devoted to the discovery of this overlooked metabolic pathway in bacteria and plants. In this second issue, the state of the art on the knowledge of the genes and enzymes of the MVA-independent MEP pathway is presented, with perspectives toward the development of new antimicrobial drugs and the regulation of isoprenoid biosynthesis in plants.

Flavonoids: true or promiscuous inhibitors of enzyme? The case of deoxyxylulose phosphate reductoisomerase.

Flavonoids, due to their physical and chemical properties (among them hydrophobicity and metal chelation abilities), are potential inhibitors of the 1-deoxyxylulose 5-phosphate reductoisomerase and most of the tested flavonoids effectively inhibited its activity with encouraging IC50 values in the micromolar range. The addition of 0.01% Triton X100 in the assays led however, to a dramatic decrease of the inhibition revealing that a non-specific inhibition probably takes place. Our study highlights the possibility of erroneous conclusions regarding the inhibition of enzymes by flavonoids that are able to produce aggregates in micromolar range. Therefore, the addition of a detergent in the assays prevents possible false positive hits in high throughput screenings.