Beyond the MEP Pathway: A novel kinase required for prenol utilization by malaria parasites.

A proposed treatment for malaria is a combination of fosmidomycin and clindamycin. Both compounds inhibit the methylerythritol 4-phosphate (MEP) pathway, the parasitic source of farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively). Both FPP and GGPP are crucial for the biosynthesis of several essential metabolites such as ubiquinone and dolichol, as well as for protein prenylation. Dietary prenols, such as farnesol (FOH) and geranylgeraniol (GGOH), can rescue parasites from MEP inhibitors, suggesting the existence of a missing pathway for prenol salvage via phosphorylation. In this study, we identified a gene in the genome of P. falciparum, encoding a transmembrane prenol kinase (PolK) involved in the salvage of FOH and GGOH. The enzyme was expressed in Saccharomyces cerevisiae, and its FOH/GGOH kinase activities were experimentally validated. Furthermore, conditional knockout parasites (Δ-PolK) were created to investigate the biological importance of the FOH/GGOH salvage pathway. Δ-PolK parasites were viable but displayed increased susceptibility to fosmidomycin. Their sensitivity to MEP inhibitors could not be rescued by adding prenols. Additionally, Δ-PolK parasites lost their capability to utilize prenols for protein prenylation. Experiments using culture medium supplemented with whole/delipidated human plasma in transgenic parasites revealed that human plasma has components that can diminish the effectiveness of fosmidomycin. Mass spectrometry tests indicated that both bovine supplements used in culture and human plasma contain GGOH. These findings suggest that the FOH/GGOH salvage pathway might offer an alternate source of isoprenoids for malaria parasites when de novo biosynthesis is inhibited. This study also identifies a novel kind of enzyme related to isoprenoid metabolism.

1-Deoxy-d-xylulose 5-phosphate reductoisomerase as target for anti Toxoplasma gondii agents: crystal structure, biochemical characterization and biological evaluation of inhibitors.

Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and Escherichia coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5 Šresolution revealing the inhibitor binding mode. In addition, we biologically characterize reverse α-phenyl-ß-thia and ß-oxa fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast with fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent anti-toxoplasma DXR inhibitors.

GAPDH mediates drug resistance and metabolism in Plasmodium falciparum malaria parasites.

Efforts to control the global malaria health crisis are undermined by antimalarial resistance. Identifying mechanisms of resistance will uncover the underlying biology of the Plasmodium falciparum malaria parasites that allow evasion of our most promising therapeutics and may reveal new drug targets. We utilized fosmidomycin (FSM) as a chemical inhibitor of plastidial isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway. We have thus identified an unusual metabolic regulation scheme in the malaria parasite through the essential glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Two parallel genetic screens converged on independent but functionally analogous resistance alleles in GAPDH. Metabolic profiling of FSM-resistant gapdh mutant parasites indicates that neither of these mutations disrupt overall glycolytic output. While FSM-resistant GAPDH variant proteins are catalytically active, they have reduced assembly into the homotetrameric state favored by wild-type GAPDH. Disrupted oligomerization of FSM-resistant GAPDH variant proteins is accompanied by altered enzymatic cooperativity and reduced susceptibility to inhibition by free heme. Together, our data identifies a new genetic biomarker of FSM-resistance and reveals the central role of GAPDH in MEP pathway control and antimalarial sensitivity.

Fosmidomycin transport through the phosphate-specific porins OprO and OprP of Pseudomonas aeruginosa.

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen, responsible for many hospital-acquired infections. The bacterium is quite resistant toward many antibiotics, in particular because of the fine-tuned permeability of its outer membrane (OM). General diffusion outer membrane pores are quite rare in this organism. Instead, its OM contains many substrate-specific porins. Their expression is varying according to growth conditions and virulence. Phosphate limitations, as well as pathogenicity factors, result in the induction of the two mono- and polyphosphate-specific porins, OprP and OprO, respectively, together with an inner membrane uptake mechanism and a periplasmic binding protein. These outer membrane channels could serve as outer membrane pathways for the uptake of phosphonates. Among them are not only herbicides, but also potent antibiotics, such as fosfomycin and fosmidomycin. In this study, we investigated the interaction between OprP and OprO and fosmidomycin in detail. We could demonstrate that fosmidomycin is able to bind to the phosphate-specific binding site inside the two porins. The inhibition of chloride conductance of OprP and OprO by fosmidomycin is considerably less than that of phosphate or diphosphate, but it can be measured in titration experiments of chloride conductance and also in single-channel experiments. The results suggest that fosmidomycin transport across the OM of P. aeruginosa occurs through OprP and OprO. Our data with the ones already known in the literature show that phosphonic acid-containing antibiotics are in general good candidates to treat the infections of P. aeruginosa at the very beginning through a favorable OM transport system.

The anti-virulence activity of the non-mevalonate pathway inhibitor FR900098 towards Burkholderia cenocepacia is maintained during experimental evolution.

Burkholderia cenocepacia infections are difficult to treat and there is an urgent need for alternative (combination) treatments. The use of anti-virulence therapies in combination with antibiotics is a possible strategy to increase the antimicrobial susceptibility of the pathogen and to slow down the development of resistance. In the present study we evaluated the ß-lactam and colistin-potentiating activity, and anti-virulence effect of the non-mevalonate pathway inhibitor FR900098 against B. cenocepacia in various in vitro and in vivo models. In addition, we evaluated whether repeated exposure to FR900098 alone or when combined with ceftazidime leads to increased resistance. FR900098 potentiated the activity of colistin and several ß-lactam antibiotics (aztreonam, cefepime, cefotaxime, ceftazidime, mecillinam and piperacillin) but not of imipenem and meropenem. When used alone or in combination with ceftazidime, FR900098 increased the survival of infected Galleria mellonella and Caenorhabditis elegans. Furthermore, combining ceftazidime with FR900098 resulted in a significant inhibition of the biofilm formation of B. cenocepacia. Repeated exposure to FR900098 in the C. elegans infection model did not lead to decreased activity, and the susceptibility of the evolved B. cenocepacia HI2424 lineages to ceftazidime, FR900098 and the combination of both remained unchanged. In conclusion, FR900098 reduces B. cenocepacia virulence and potentiates ceftazidime in an in vivo C. elegans model, and this activity is not lost during the experimental evolution experiment carried out in the present study.

A mevalonate bypass system facilitates elucidation of plastid biology in malaria parasites.

Malaria parasites rely on a plastid organelle for survival during the blood stages of infection. However, the entire organelle is dispensable as long as the isoprenoid precursor, isopentenyl pyrophosphate (IPP), is supplemented in the culture medium. We engineered parasites to produce isoprenoid precursors from a mevalonate-dependent pathway, creating a parasite line that replicates normally after the loss of the apicoplast organelle. We show that carbon-labeled mevalonate is specifically incorporated into isoprenoid products, opening new avenues for researching this essential class of metabolites in malaria parasites. We also show that essential apicoplast proteins, such as the enzyme target of the drug fosmidomycin, can be deleted in this mevalonate bypass parasite line, providing a new method to determine the roles of other important apicoplast-resident proteins. Several antibacterial drugs kill malaria parasites by targeting basic processes, such as transcription, in the organelle. We used metabolomic and transcriptomic methods to characterize parasite metabolism after azithromycin treatment triggered loss of the apicoplast and found that parasite metabolism and the production of apicoplast proteins is largely unaltered. These results provide insight into the effects of apicoplast-disrupting drugs, several of which have been used to treat malaria infections in humans. Overall, the mevalonate bypass system provides a way to probe essential aspects of apicoplast biology and study the effects of drugs that target apicoplast processes.

Metabolic Survival Adaptations of Plasmodium falciparum Exposed to Sublethal Doses of Fosmidomycin.

The malaria parasite Plasmodium falciparum contains the apicoplast organelle that synthesizes isoprenoids, which are metabolites necessary for posttranslational modification of Plasmodium proteins. We used fosmidomycin, an antibiotic that inhibits isoprenoid biosynthesis, to identify mechanisms that underlie the development of the parasite's adaptation to the drug at sublethal concentrations. We first determined a concentration of fosmidomycin that reduced parasite growth by ∼50% over one intraerythrocytic developmental cycle (IDC). At this dose, we maintained synchronous parasite cultures for one full IDC and collected metabolomic and transcriptomic data at multiple time points to capture global and stage-specific alterations. We integrated the data with a genome-scale metabolic model of P. falciparum to characterize the metabolic adaptations of the parasite in response to fosmidomycin treatment. Our simulations showed that, in treated parasites, the synthesis of purine-based nucleotides increased, whereas the synthesis of phosphatidylcholine during the trophozoite and schizont stages decreased. Specifically, the increased polyamine synthesis led to increased nucleotide synthesis, while the reduced methyl-group cycling led to reduced phospholipid synthesis and methyltransferase activities. These results indicate that fosmidomycin-treated parasites compensate for the loss of prenylation modifications by directly altering processes that affect nucleotide synthesis and ribosomal biogenesis to control the rate of RNA translation during the IDC. This also suggests that combination therapies with antibiotics that target the compensatory response of the parasite, such as nucleotide synthesis or ribosomal biogenesis, may be more effective than treating the parasite with fosmidomycin alone.

Fosmidomycin, an inhibitor of isoprenoid synthesis, induces persistence in Chlamydia by inhibiting peptidoglycan assembly.

The antibiotic, fosmidomycin (FSM) targets the methylerythritol phosphate (MEP) pathway of isoprenoid synthesis by inhibiting the essential enzyme, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) and is lethal to intracellular parasites and bacteria. The obligate intracellular bacterial pathogen, Chlamydia trachomatis, alternates between two developmental forms: the extracellular, infectious elementary body (EB), and the intracellular, replicative form called the reticulate body (RB). Several stressful growth conditions including iron deprivation halt chlamydial cell division and cause development of a morphologically enlarged, but viable form termed an aberrant body (AB). This phenotype constitutes the chlamydial developmental state known as persistence. This state is reversible as removal of the stressor allows the chlamydiae to re-enter and complete the normal developmental cycle. Bioinformatic analysis indicates that C. trachomatis encodes a homolog of Dxr, but its function and the requirement for isoprenoid synthesis in chlamydial development is not fully understood. We hypothesized that chlamydial Dxr (DxrCT) is functional and that the methylerythritol phosphate (MEP) pathway is required for normal chlamydial development. Thus, FSM exposure should be lethal to C. trachomatis. Overexpression of chlamydial Dxr (DxrCT) in Escherichia coli under FSM exposure and in a conditionally lethal dxr mutant demonstrated that DxrCT functions similarly to E. coli Dxr. When Chlamydia-infected cultures were exposed to FSM, EB production was significantly reduced. However, titer recovery assays, electron microscopy, and peptidoglycan labeling revealed that FSM inhibition of isoprenoid synthesis is not lethal to C. trachomatis, but instead induces persistence. Bactoprenol is a critical isoprenoid required for peptidoglycan precursor assembly. We therefore conclude that FSM induces persistence in Chlamydia by preventing bactoprenol production necessary for peptidoglycan precursor assembly and subsequent cell division.

Fosmidomycin biosynthesis diverges from related phosphonate natural products.

Fosmidomycin and related molecules comprise a family of phosphonate natural products with potent antibacterial, antimalarial and herbicidal activities. To understand the biosynthesis of these compounds, we characterized the fosmidomycin producer, Streptomyces lavendulae, using biochemical and genetic approaches. We were unable to elicit production of fosmidomycin, instead observing the unsaturated derivative dehydrofosmidomycin, which we showed potently inhibits 1-deoxy-D-xylulose-5-phosphate reductoisomerase and has bioactivity against a number of bacteria. The genes required for dehydrofosmidomycin biosynthesis were established by heterologous expression experiments. Bioinformatics analyses, characterization of intermediates and in vitro biochemistry show that the biosynthetic pathway involves conversion of a two-carbon phosphonate precursor into the unsaturated three-carbon product via a highly unusual rearrangement reaction, catalyzed by the 2-oxoglutarate dependent dioxygenase DfmD. The required genes and biosynthetic pathway for dehydrofosmidomycin differ substantially from that of the related natural product FR-900098, suggesting that the ability to produce these bioactive molecules arose via convergent evolution.

α,α-Difluorophosphonohydroxamic Acid Derivatives among the Best Antibacterial Fosmidomycin Analogues.

Three α,α-difluorophosphonate derivatives of fosmidomycin were synthesized from diethyl 1,1-difluorobut-3-enylphosphonate and were evaluated on Escherichia coli. Two of them are among the best 1-deoxy-d-xylulose 5-phosphate reductoisomerase inhibitors, with IC50 in the nM range, much better than fosmidomycin, the reference compound. They also showed an enhanced antimicrobial activity against E. coli on Petri dishes in comparison with the corresponding phosphates and the non-fluorinated phosphonate.

Synthesis and anti-parasitic activity of N-benzylated phosphoramidate Mg2+-chelating ligands.

A series of N-benzylated phosphoramidate esters, containing a 3,4-dihydroxyphenyl Mg2+-chelating group, has been synthesised in five steps as analogues of fosmidomycin, a Plasmodium falciparum 1-deoxy-1-d-xylulose-5-phosphate reductoisomerase (PfDXR) inhibitor. The 3,4-dihydroxyphenyl group effectively replaces the Mg2+-chelating hydroxamic acid group in fosmidomycin. The compounds showed very encouraging anti-parasitic activity with IC50 values of 5.6-16.4 µM against Plasmodium falciparum parasites and IC50 values of 5.2 - 10.2 µM against Trypanosoma brucei brucei (T.b.brucei). Data obtained from in silico docking of the ligands in the PfDXR receptor cavity (3AU9)5 support their potential as PfDXR inhibitors.

Synthesis and Antiplasmodial Activity of Novel Fosmidomycin Derivatives and Conjugates with Artemisinin and Aminochloroquinoline.

Malaria, despite many efforts, remains among the most problematic infectious diseases worldwide, mainly due to the development of drug resistance by Plasmodium falciparum. The antibiotic fosmidomycin (FSM) is also known for its antimalarial activity by targeting the non-mevalonate isoprenoid synthesis pathway, which is essential for the malaria parasites but is absent in mammalians. In this study, we synthesized and evaluated against the chloroquine-resistant P. falciparum FcB1/Colombia strain, a series of FSM analogs, derivatives, and conjugates with other antimalarial agents, such as artemisinin (ART) and aminochloroquinoline (ACQ). The biological evaluation revealed four new compounds with higher antimalarial activity than FSM: two FSM-ACQ derivatives and two FSM-ART conjugates, with 3.5-5.4 and 41.5-23.1 times more potent activities than FSM, respectively.

Predicting Drug Resistance Using Deep Mutational Scanning.

Drug resistance is a major healthcare challenge, resulting in a continuous need to develop new inhibitors. The development of these inhibitors requires an understanding of the mechanisms of resistance for a critical mass of occurrences. Recent genome editing technologies based on high-throughput DNA synthesis and sequencing may help to predict mutations resulting in resistance by testing large mutagenesis libraries. Here we describe the rationale of this approach, with examples and relevance to drug development and resistance in malaria.

Double prodrugs of a fosmidomycin surrogate as antimalarial and antitubercular agents.

A series of eleven double prodrug derivatives of a fosmidomycin surrogate were synthesized and investigated for their ability to inhibit in vitro growth of P. falciparum and M. tuberculosis. A pivaloyloxymethyl (POM) phosphonate prodrug modification was combined with various prodrug derivatisations of the hydroxamate moiety. The majority of compounds showed activity comparable with or inferior to fosmidomycin against P. falciparum. N-benzyl substituted carbamate prodrug 6f was the most active antimalarial analog with an IC50 value of 0.64 µM. Contrary to fosmidomycin and parent POM-prodrug 5, 2-nitrofuran and 2-nitrothiophene prodrugs 6i and 6j displayed promising antitubercular activities.

Phosphonodiamidate prodrugs of N-alkoxy analogs of a fosmidomycin surrogate as antimalarial and antitubercular agents.

A series of N-alkoxy analogs of a l-leucine ethyl ester phosphonodiamidate prodrug of a fosmidomycin surrogate were synthesized and investigated for their ability to inhibit in vitro growth of P. falciparum and M. tuberculosis. These compounds originate by merging a previously reported successful phosphonate derivatisation with favorable modifications of the hydroxamate moiety. None of the synthesized compounds showed enhanced activity against either P. falciparum or M. tuberculosis in comparison with the parent free hydroxamate analog.

Characterization of the flavin monooxygenase involved in biosynthesis of the antimalarial FR-900098.

The latter steps in this biosynthetic pathway for the antimalarial phosphonic acid FR-900098 include the installation of a hydroxamate onto 3-aminopropylphosphonate, which is catalyzed by the consecutive actions of an acetyltransferase and an amine hydroxylase. Here, we present the 1.6 Å resolution co-crystal structure and accompanying biochemical characterization of FrbG, which catalyzes the hydroxylation of aminopropylphosphonate. We show that FrbG is a flavin-dependent N-hydroxylating monooxygenase (NMO), which shares a similar overall structure with flavin-containing monooxygenases (FMOs). Notably, we also show that the cytidine-5'-monophosphate moiety of the substrate is a critical determinant of specificity, distinguishing FrbG from other FMOs in that the nucleotide cofactor-binding domain also serves in conferring substrate recognition. In the FrbG-FAD+-NADPH co-crystal structure, the C4 of the NADPH nicotinamide is situated near the N5 of the FAD isoalloxazine, and is oriented with a distance and stereochemistry to facilitate hydride transfer.

A computational study of the molecular basis of antibiotic resistance in a DXR mutant.

Proteins of the independent mevalonate pathway for isoprenoid biosynthesis are important targets for the development of new antibacterial compounds as this pathway is present in most pathogenic organisms such as Mycobacterium tuberculosis, DPlasmodium falciparum and Escherichia coli, but is not present in mammalian species, including humans. Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is an important target in this pathway and the most effective DXR inhibitor to date is fosmidomycin, which is used to treat malaria and, more recently, tuberculosis. Recently, Armstrong C. M. et al. showed that a mutant of DXR, S222T, induces a loss of the fosmidomycin inhibition efficiency, even though the bacteria culture is still viable and able to produce isoprenoids. As this represents a potential fosmidomycin-resistant mutation, it is important to understand the mechanism of this apparent mutation-induced resistance to fosmidomycin. Here, we used molecular dynamics simulations and Molecular Mechanics/Poisson Boltzmann Surface Area analysis to understand the structural and energetic basis of the resistance. Our results suggest that the point mutation results in changes to the structural dynamics of an active site loop that probably protects the active site and facilitates enzymatic reaction. From the simulation analysis, we also showed that the mutation results in changes in the interaction energy profiles in a way that can explain the observed activity of the mutant protein toward the natural inhibitor deoxy-D-xylulose 5-phosphate. These results should be taken into consideration in future efforts to develop new therapeutic antibiotic compounds that target DXR.

Amino acid based prodrugs of a fosmidomycin surrogate as antimalarial and antitubercular agents.

Fosmidomycin is a natural antibiotic with promising IspC (DXR, 1-deoxy-d-xylulose-5-phosphate reductoisomerase) inhibitory activity. This enzyme catalyzes the first committed step of the non-mevalonate isoprenoid biosynthesis pathway, which is essential in Plasmodium falciparum and Mycobacterium tuberculosis. Mainly as a result of its high polarity, fosmidomycin displays suboptimal pharmacokinetic properties. Furthermore, fosmidomycin is inactive against M. tuberculosis as a result of its inability to penetrate the bacterial cell wall. Temporarily masking the phosphonate moiety as a prodrug has the potential to solve both issues. We report the application of two amino acid based prodrug approaches on a fosmidomycin surrogate. Conversion of the phosphonate moiety into tyrosine-derived esters increases the in vitro activity against asexual blood stages of P. falciparum, while phosphonodiamidate prodrugs display promising antitubercular activities. Selected prodrugs were tested in vivo in a P. berghei malaria mouse model. These results indicate good in vivo antiplasmodial potential.

Efficacy and Safety of Fosmidomycin-Piperaquine as Nonartemisinin-Based Combination Therapy for Uncomplicated Falciparum Malaria: A Single-Arm, Age De-escalation Proof-of-Concept Study in Gabon.

Background: Fosmidomycin-piperaquine is being developed as nonartemisinin-based combination therapy to meet the challenge of emerging artemisinin resistance. Methods: The study was a phase 2, single-arm, proof-of-concept study of the efficacy, tolerability, and safety of fosmidomycin-piperaquine for the treatment of uncomplicated Plasmodium falciparum monoinfection in Gabon. Adults and children of both sexes with initial parasite counts between 1000 and 150000/µL received oral treatment with fosmidomycin (twice daily doses of 30 mg/kg) and piperaquine (once daily dose of 16 mg/kg) for 3 days and followed-up for 63 days. The primary efficacy endpoint was the per-protocol polymerase chain reaction (PCR)-corrected day 28 adequate clinical and parasitological response (ACPR). Results: One hundred patients were enrolled. The PCR-corrected day 28 ACPR rate was 83/83, or 100% (95% confidence interval, 96-100). Fourteen patients had asexual parasitaemia between day 28 and day 63; all were typed by PCR as new infections. Fosmidomycin-piperaquine therapy led to rapid parasite clearance (median, 36 hours; interquartile range [IQR], 6-60) and fever clearance time (median, 12 hours; IQR, 6-48). The electrocardiogram assessments showed 2 patients with prolonged QT interval >500 msec following study drug administration. The majority of adverse events affected the gastrointestinal and respiratory tracts and were transient and mild to moderate in severity. Conclusions: This is the first report of the use of the combination fosmidomycin-piperaquine. The combination appeared to have high efficacy and be safe and well tolerated despite observed transient changes in electrocardiogram with prolongation of the QT interval. Clinical Trials Registration. NCT02198807.

Spectacular Oscillations in Plant Isoprene Emission under Transient Conditions Explain the Enigmatic CO2 Response.

Plant isoprene emissions respond to light and temperature similarly to photosynthesis, but CO2 dependencies of isoprene emission and photosynthesis are profoundly different, with photosynthesis increasing and isoprene emission decreasing with increasing CO2 concentration due to reasons not yet understood. We studied isoprene emission, net assimilation rate, and chlorophyll fluorescence under different CO2 and O2 concentrations in the strong isoprene emitter hybrid aspen (Populus tremula × Populus tremuloides), and used rapid changes in ambient CO2 or O2 concentrations or light level to induce oscillations. As isoprene-emitting species support very high steady-state chloroplastic pool sizes of the primary isoprene substrate, dimethylallyl diphosphate (DMADP), which can mask the effects of oscillatory dynamics on isoprene emission, the size of the DMADP pool was experimentally reduced by either partial inhibition of isoprenoid synthesis pathway by fosmidomycin-feeding or by changes in ambient gas concentrations leading to DMADP pool depletion in intact leaves. In feedback-limited conditions observed at low O2 and/or high CO2 concentration under which the rate of photosynthesis is governed by the limited rate of ATP and NADPH formation due to low chloroplastic phosphate levels, oscillations in photosynthesis and isoprene emission were repeatedly induced by rapid environmental modifications in both partly fosmidomycin-inhibited leaves and in intact leaves with in vivo reduced DMADP pools. The oscillations in net assimilation rate and isoprene emission in feedback-inhibited leaves were in the same phase, and relative changes in the pools of photosynthetic metabolites and DMADP estimated by in vivo kinetic methods were directly proportional through all oscillations induced by different environmental perturbations. We conclude that the oscillations in isoprene emission provide direct experimental evidence demonstrating that the response of isoprene emission to changes in ambient gas concentrations is controlled by the chloroplastic reductant supply.