Isopentenyl Phosphate Research Articles

The utility of Streptococcus mutans undecaprenol kinase for the chemoenzymatic synthesis of diverse non-natural isoprenoids

Isoprene chemoenzymatic cascades (ICCs) overcome the complexity of natural pathways by leveraging a streamlined two-enzyme cascade, facilitating efficient synthesis of C5-isoprene diphosphate precursors from readily available alcohol derivatives. Despite the documented promiscuity of enzymes in ICCs, exploration of their potential for accessing novel compounds remains limited, and existing methods require additional enzymes for generating longer-chain diphosphates. In this study, we present the utility of Streptococcus mutans undecaprenol kinase (SmUdpK) for the chemoenzymatic synthesis of diverse non-natural isoprenoids. Using a library of 50 synthetic alcohols, we demonstrate that SmUdpK’s promiscuity extends to allylic chains as small as four carbons and benzylic alcohols with various substituents. Subsequently, SmUdpK is utilized in an ICC with isopentenyl phosphate kinase and aromatic prenyltransferase to generate multiple non-natural isoprenoids. This work provides evidence that, with proper optimization, SmUdpK can act as the first enzyme in these ICCs, enhancing access to both valuable and novel compounds.

Isoprene emission dynamics in Chaetoceros curvisetus: Insights from transcriptome analysis under light/dark cycles

Isoprene from marine emissions significantly influences the atmospheric oxidative capacity and secondary pollutants in the marine boundary layer. While marine algae are recognized as primary contributors to marine emissions of isoprene, the long-term and dark-phase isoprene production from these organisms remains underexplored, potentially lead to inaccuracies in estimating marine isoprene emissions. In this study, we conducted a time series investigation of isoprene emission from Chaetoceros curvisetus with a growth cycle duration of 19 days and examined the influence of environmental factors. Our findings revealed that C. curvisetus emits isoprene during its whole growth cycle, with peak emissions (2.82 × 10−11 μmol cells−1 h−1) recorded in the stationary phase. Under nitrogen, light and temperature stress, significant release of isoprene is found. During both the light and dark phases, isoprene emission was observed with comparable intensities. Transcriptome analysis showed that 41 differentially expressed transcripts were involved in the synthesis of volatile organic compounds. Notably, downregulation of genes associated with pyruvate synthesis in the glycolysis pathway suggests its importance for dark isoprene release. The study also highlighted downregulation of isopentenyl phosphate kinase and Gene Ontology enrichment in organelles housing mevalonate (MVA) pathways, suggesting that exchange involving the transport of intermediate metabolites between MVA and methylerythritol phosphate (MEP) pathway may contribute to the released isoprene in dark phase. Furthermore, the synthesis of substrates and downstream products is related to the dark release of isoprene. In conclusion, our study illuminates the pivotal roles of substrate availability, interplay between MVA and MEP pathways, and glycolysis pathway in governing isoprene emissions under light/dark cycles.

Ternary complexes of isopentenyl phosphate kinase from Thermococcus paralvinellae reveal molecular determinants of non-natural substrate specificity.

Isopentenyl phosphate kinases (IPKs) have recently garnered attention for their central role in biocatalytic "isoprenol pathways," which seek to reduce the synthesis of the isoprenoid precursors to two enzymatic steps. Furthermore, the natural promiscuity of IPKs toward non-natural alkyl-monophosphates (alkyl-Ps) as substrates has hinted at the isoprenol pathways' potential to access novel isoprenoids with potentially useful activities. However, only a handful of IPK crystal structures have been solved to date, and even fewer of these contain non-natural substrates bound in the active site. The current study sought to elucidate additional ternary complexes bound to non-natural substrates using the IPK homolog from Thermococcus paralvinellae (TcpIPK). Four such structures were solved, each bound to a different non-natural alkyl-P and the phosphoryl donor substrate/product adenosine triphosphate (ATP)/adenosine diphosphate (ADP). As expected, the quaternary, tertiary, and secondary structures of TcpIPK closely resembled those of IPKs published previously, and kinetic analysis of a novel alkyl-P substrate highlighted the potentially dramatic effects of altering the core scaffold of the natural substrate. Even more interesting, though, was the discovery of a trend correlating the position of two α helices in the active site with the magnitude of an IPK homolog's reaction rate for the natural reaction. Overall, the current structures of TcpIPK highlight the importance of continued structural analysis of the IPKs to better understand and optimize their activity with both natural and non-natural substrates.

Functional Characterization and Screening of Promiscuous Kinases and Isopentenyl Phosphate Kinases for the Synthesis of DMAPP via a One-Pot Enzymatic Cascade.

Dimethylallyl diphosphate (DMAPP) is a key intermediate metabolite in the synthesis of isoprenoids and is also the prenyl donor for biosynthesizing prenylated flavonoids. However, it is difficult to prepare DMAPP via chemical and enzymatic methods. In this study, three promiscuous kinases from Shigella flexneri (SfPK), Escherichia coli (EcPK), and Saccharomyces cerevisiae (ScPK) and three isopentenyl phosphate kinases from Methanolobus tindarius (MtIPK), Methanothermobacter thermautotrophicus str. Delta H (MthIPK), and Arabidopsis thaliana (AtIPK) were cloned and expressed in Escherichia coli. The enzymatic properties of recombinant enzymes were determined. The Kcat/Km value of SfPK for DMA was 6875 s-1 M-1, which was significantly higher than those of EcPK and ScPK. The Kcat/Km value of MtIPK for DMAP was 402.9 s-1 M-1, which was ~400% of that of MthIPK. SfPK was stable at pH 7.0-9.5 and had a 1 h half-life at 65 °C. MtIPK was stable at pH 6.0-8.5 and had a 1 h half-life at 50 °C. The stability of SfPK and MtIPK was better than that of the other enzymes. Thus, SfPK and MtIPK were chosen to develop a one-pot enzymatic cascade for producing DMAPP from DMA because of their catalytic efficiency and stability. The optimal ratio between SfPK and MtIPK was 1:8. The optimal pH and temperature for the one-pot enzymatic cascade were 7.0 and 35 °C, respectively. The optimal concentrations of ATP and DMA were 10 and 80 mM, respectively. Finally, maximum DMAPP production reached 1.23 mM at 1 h under optimal conditions. Therefore, the enzymatic method described herein for the biosynthesis of DMAPP from DMA can be widely used for the synthesis of isoprenoids and prenylated flavonoids.

Engineering Escherichia coli for effective and economic production of cis-abienol by optimizing isopentenol utilization pathway

Efficient and economical supply of cis-abienol has attracted extensive attention due to its use as a precursor in the synthesis of ambrox (a cardinal odor of rare ambergris). Increasing awareness in environmental issues and advantages of microbial production–sustainable and green manufacture, have motivated efforts to advance the cis-abienol biosynthesis in more affordable ways. Herein, Escherichia coli cells were employed to scale up cis-abienol production via expressing the concise isopentenol utilization pathway (IUP). Alcohol kinases and isopentenyl phosphate kinases (IPKs) were screened and coordinated to increase dimethylallyl diphosphate (DMAPP) supply, which resulted in approximately 37-fold improvements in the cis-abienol yield, compared with the titer obtained by endogenous MEP pathway. Next, we constructed a double-phosphatase-deficient strain (BD203), resulting in a 40% increase in the cis-abienol titer relative to wild-type strains. By optimization of addition strategies, a cis-abienol titer of 311.8 ± 1.0 mg/L was obtained in strain BD203, which is the highest cis-abienol titer reported in shake flasks and 14.7% of the maximum theoretical yield. Finally, further optimization of the fed-batch fermentation process enabled a cis-abienol titer of 1375.7 mg/L in a 1.3 L bioreactor. Overall, BD203 achieved a substantial increase in the cis-abienol titer in this study and engineering microbes were expected to facilitate progress toward industrial production.

Diverting mevalonate pathway metabolic flux leakage in Saccharomyces cerevisiae for monoterpene geraniol production from cane molasses

Geraniol has versatile applications in the fragrance, pharmacy, and biofuel industries. This monoterpene is produced via the mevalonate pathway in the yeast Saccharomyces cerevisiae. However, carbon flux leakage restricts the efficient biosynthesis of target compounds via this pathway. In this work, the mevalonate pathway of the recombinant S. cerevisiae was reprogrammed by introducing an isopentenyl phosphate kinase (IPK) and to establish an isopentenyl phosphate utilization (IPU) bypass that can utilise the isopentenyl phosphate (IP) produced from phosphomevalonate decarboxylase (PMD). Geraniol production was increased by 225% in the engineered strain. The yield of geraniol was further enhanced to 156.2 ± 6.6 mg/L through optimisation of key rate-limiting enzymes of the mevalonate pathway. Using pretreated molasses as a carbon source yielded 180.9 ± 2.9 mg/L of geraniol under optimal fermentation conditions, the highest reported titre to date using molasses medium. Modification of the mevalonate pathway can provide a commercially feasible strategy for monoterpene synthesis in engineered S. cerevisiae.

Molecular Basis for the Substrate Promiscuity of Isopentenyl Phosphate Kinase from Candidatus methanomethylophilus alvus.

Isopentenyl phosphate kinases (IPKs) catalyze the ATP-dependent phosphorylation of isopentenyl monophosphate (IP) to isopentenyl diphosphate (IPP) in the alternate mevalonate pathways of the archaea and plant cytoplasm. In recent years, IPKs have also been employed in artificial biosynthetic pathways called "(iso) prenol pathways" that utilize promiscuous kinases to sequentially phosphorylate (iso) prenol and generate the isoprenoid precursors IPP and dimethylallyl diphosphate (DMAPP). Furthermore, IPKs have garnered attention for their impressive substrate promiscuity toward non-natural alkyl-monophosphates (alkyl-Ps), which has prompted their utilization as biocatalysts for the generation of novel isoprenoids. However, none of the IPK crystal structures currently available contain non-natural substrates, leaving the roles of active-site residues in substrate promiscuity ambiguous. To address this, we present herein the high-resolution crystal structures of an IPK from Candidatus methanomethylophilus alvus (CMA) in the apo form and bound to natural and non-natural substrates. Additionally, we describe active-site engineering studies leading to enzyme variants with broadened substrate scope, as well as structure determination of two such variants (Ile74Ala and Ile146Ala) bound to non-natural alkyl-Ps. Collectively, our crystallographic studies compare six structures of CMA variants in different ligand-bound forms and highlight contrasting structural dynamics of the two substrate-binding sites. Furthermore, the structural and mutational studies confirm a novel role of the highly conserved DVTGG motif in catalysis, both in CMA and in IPKs at large. As such, the current study provides a molecular basis for the substrate-binding modes and catalytic performance of CMA toward the goal of developing IPKs into useful biocatalysts.

Biochemical Characterization of a Novel Prenyltransferase from Streptomyces sp. NT11 and Development of a Recombinant Strain for the Production of 6-Prenylnaringenin.

Prenyl groups increase the lipophilicity of flavonoids, endowing them with a special activity, selectivity, and pharmacological properties by prenylation. Herein, a novel prenyltransferase (ShFPT) gene from Streptomyces sp. NT11 was expressed in Escherichia coli, and its biochemical characteristics were determined. ShFPT exhibited high selectivity to prenylate naringenin at C-6 to generate 6-prenylnaringenin. The optimal activity was observed at pH 6.0 and 55 °C. The Kcat and Km for naringenin were 0.0095 s-1 and 0.20 mM, respectively. Several promiscuous kinase and isopentenyl phosphate kinase genes were screened to develop the most efficient dimethylallyl diphosphate (DMAPP) synthesis pathway for 6-prenylnaringenin synthesis in E. coli. The 6-prenylnaringenin production was improved by changing the induction strategies and optimizing the bioconversion conditions. Finally, 6-prenylnaringenin production reached the highest yield of 69.9 mg/L with average productivity of 4.0 mg/L/h after 16 h incubation, which is the highest yield for any prenylated flavonoid reported to date in E. coli. Therefore, this study provides an efficient method for 6-prenylnaringenin production and reveals the DMAPP synthesis pathway.

Novel Homologs of Isopentenyl Phosphate Kinase Reveal Class-Wide Substrate Flexibility.

The widespread utility of isoprenoids has recently sparked interest in efficient synthesis of isoprene‐diphosphate precursors. Current efforts have focused on evaluating two‐step “isoprenol pathways,” which phosphorylate prenyl alcohols using promiscuous kinases/phosphatases. The convergence on isopentenyl phosphate kinases (IPKs) in these schemes has prompted further speculation about the class's utility in synthesizing non‐natural isoprenoids. However, the substrate promiscuity of IPKs in general has been largely unexplored. Towards this goal, we report the biochemical characterization of five novel IPKs from Archaea and the assessment of their substrate specificity using 58 alkyl‐monophosphates. This study reveals the IPK‐catalyzed synthesis of 38 alkyl‐diphosphate analogs and discloses broad substrate specificity of IPKs. Further, to demonstrate the biocatalytic utility of IPK‐generated alkyl‐diphosphates, we also highlight the synthesis of alkyl‐l‐tryptophan derivatives using coupled IPK‐prenyltransferase reactions. These results reveal IPK‐catalyzed reactions are compatible with downstream isoprenoid enzymes and further support their development as biocatalytic tools for the synthesis of non‐natural isoprenoids.

Overcoming Bottlenecks for Metabolic Engineering of Sesquiterpene Production in Tomato Fruits.

Terpenoids are a large and diverse class of plant metabolites that also includes volatile mono- and sesquiterpenes which are involved in biotic interactions of plants. Due to the limited natural availability of these terpenes and the tight regulation of their biosynthesis, there is strong interest to introduce or enhance their production in crop plants by metabolic engineering for agricultural, pharmaceutical and industrial applications. While engineering of monoterpenes has been quite successful, expression of sesquiterpene synthases in engineered plants frequently resulted in production of only minor amounts of sesquiterpenes. To identify bottlenecks for sesquiterpene engineering in plants, we have used two nearly identical terpene synthases, snapdragon (Antirrhinum majus) nerolidol/linalool synthase-1 and -2 (AmNES/LIS-1/-2), that are localized in the cytosol and plastids, respectively. Since these two bifunctional terpene synthases have very similar catalytic properties with geranyl diphosphate (GPP) and farnesyl diphosphate (FPP), their expression in target tissues allows indirect determination of the availability of these substrates in both subcellular compartments. Both terpene synthases were expressed under control of the ripening specific PG promoter in tomato fruits, which are characterized by a highly active terpenoid metabolism providing precursors for carotenoid biosynthesis. As AmNES/LIS-2 fruits produced the monoterpene linalool, AmNES/LIS-1 fruits were found to exclusively produce the sesquiterpene nerolidol. While nerolidol emission in AmNES/LIS-1 fruits was 60- to 584-fold lower compared to linalool emission in AmNES/LIS-2 fruits, accumulation of nerolidol-glucosides in AmNES/LIS-1 fruits was 4- to 14-fold lower than that of linalool-glucosides in AmNES/LIS-2 fruits. These results suggest that only a relatively small pool of FPP is available for sesquiterpene formation in the cytosol. To potentially overcome limitations in sesquiterpene production, we transiently co-expressed the key pathway-enzymes hydroxymethylglutaryl-CoA reductase (HMGR) and 1-deoxy-D-xylulose 5-phosphate synthase (DXS), as well as the regulator isopentenyl phosphate kinase (IPK). While HMGR and IPK expression increased metabolic flux toward nerolidol formation 5.7- and 2.9-fold, respectively, DXS expression only resulted in a 2.5-fold increase.

Cover Feature: Probing the Substrate Promiscuity of Isopentenyl Phosphate Kinase as a Platform for Hemiterpene Analogue Production (ChemBioChem 17/2019)

Cover Feature: Probing the Substrate Promiscuity of Isopentenyl Phosphate Kinase as a Platform for Hemiterpene Analogue Production (ChemBioChem 17/2019)

Probing the Substrate Promiscuity of Isopentenyl Phosphate Kinase as a Platform for Hemiterpene Analogue Production.

Isoprenoids are a large class of natural products with wide-ranging applications. Synthetic biology approaches to the manufacture of isoprenoids and their new-to-nature derivatives are limited due to the provision in nature of just two hemiterpene building blocks for isoprenoid biosynthesis. To address this limitation, artificial chemo-enzymatic pathways such as the alcohol-dependent hemiterpene (ADH) pathway serve to leverage consecutive kinases to convert exogenous alcohols into pyrophosphates that could be coupled to downstream isoprenoid biosynthesis. To be successful, each kinase in this pathway should be permissive of a broad range of substrates. For the first time, we have probed the promiscuity of the second enzyme in the ADH pathway-isopentenyl phosphate kinase from Thermoplasma acidophilum-towards a broad range of acceptor monophosphates. Subsequently, we evaluate the suitability of this enzyme to provide unnatural pyrophosphates and provide a critical first step in characterizing the rate-limiting steps in the artificial ADH pathway.

Substrate Specificity and Engineering of Mevalonate 5-Phosphate Decarboxylase.

A bifurcation of the mevalonate (MVA) pathway was recently discovered in bacteria of the Chloroflexi phylum. In this alternative route for the biosynthesis of isopentenylpyrophosphate (IPP), the penultimate step is the decarboxylation of (R)-mevalonate 5-phosphate ((R)-MVAP) to isopentenyl phosphate (IP), which is followed by the ATP-dependent phosphorylation of IP to IPP catalyzed by isopentenyl phosphate kinase (IPK). Notably, the decarboxylation reaction is catalyzed by mevalonate 5-phosphate decarboxylase (MPD), which shares considerable sequence similarity with mevalonate diphosphate decarboxylase (MDD) of the classical MVA pathway. We show that an enzyme originally annotated as an MDD from the Chloroflexi bacterium Anaerolinea thermophila possesses equal catalytic efficiency for (R)-MVAP and (R)-mevalonate 5-diphosphate ((R)-MVAPP). Further, the molecular basis for this dual specificity is revealed by near atomic-resolution X-ray crystal structures of A. thermophila MPD/MDD bound to (R)-MVAP or (R)-MVAPP. These findings, when combined with sequence and structural comparisons of this bacterial enzyme, functional MDDs, and several putative MPDs, delineate key active-site residues that confer substrate specificity and functionally distinguish MPD and MDD enzyme classes. Extensive sequence analyses identified functional MPDs in the halobacteria class of archaea that had been annotated as MDDs. Finally, no eukaryotic MPD candidates were identified, suggesting the absence of the alternative MVA (altMVA) pathway in all eukaryotes, including, paradoxically, plants, which universally encode a structural and functional homologue of IPK. Additionally, we have developed a viable engineered strain of Saccharomyces cerevisiae as an in vivo metabolic model and a synthetic biology platform for enzyme engineering and terpene biosynthesis in which the classical MVA pathway has been replaced with the altMVA pathway.

Exploring natural biodiversity to expand access to microbial terpene synthesis

BackgroundTerpenes are industrially relevant natural compounds the biosynthesis of which relies on two well-established—mevalonic acid (MVA) and methyl erythritol phosphate (MEP)-pathways. Both pathways are widely distributed in all domains of life, the former is predominantly found in eukaryotes and archaea and the latter in eubacteria and chloroplasts. These two pathways supply isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the universal building blocks of terpenes.ResultsThe potential to establish a semisynthetic third pathway to access these precursors has been investigated in the present work. We have tested the ability of a collection of 93 isopentenyl phosphate kinases (IPK) from the biodiversity to catalyse the double phosphorylation of isopentenol and dimethylallyl alcohol to give, respectively IPP and DMAPP. Five IPKs selected from a preliminary in vitro screening were evaluated in vivo in an engineered chassis E. coli strain producing carotenoids. The recombinant pathway leading to the synthesis of neurosporene and lycopene, allows a simple colorimetric assay to test the potential of IPKs for the synthesis of IPP and DMAPP starting from the corresponding alcohols. The best candidate identified was the IPK from Methanococcoides burtonii (UniProt ID: Q12TH9) which improved carotenoid and neurosporene yields ~ 18-fold and > 45-fold, respectively. In our lab scale conditions, titres of neurosporene reached up to 702.1 ± 44.7 µg/g DCW and 966.2 ± 61.6 µg/L. A scale up to 4 L in-batch cultures reached to 604.8 ± 68.3 µg/g DCW and 430.5 ± 48.6 µg/L without any optimisation shown its potential for future applications. Neurosporene was almost the only carotenoid produced under these conditions, reaching ~ 90% of total carotenoids both at lab and batch scales thus offering an easy access to this sophisticated molecule.ConclusionIPK biodiversity was screened in order to identify IPKs that optimize the final carotenoid content of engineered E. coli cells expressing the lycopene biosynthesis pathway. By simply changing the IPK and without any other metabolic engineering we improved the neurosporene content by more than 45 fold offering a new biosynthetic access to this molecule of upmost importance.

Substrate Specificity of an Isopentenyl Phosphate Kinase

Isopentenyl diphosphate and dimethylallyl diphosphate are the fundamental biosynthetic precursors for >80,000 diverse isoprenoids including NPs, cholesterol, coenzyme Q, chlorophyll, and vitamin A, of all domain of life. These precursors are biosynthesized via methylerythritol phosphate (MEP) pathway in bacteria and mevalonate (MVA) pathway in eukaryotes, archaea, some eubacteria. Recently, an alternate MVA pathway has been identified in archaea and plants, that involves phosphorylation of isopentenyl monophosphate by isopentenyl phosphate kinase (IPK) to yield isopentenyl diphosphate. In this poster we will demonstrate IPK to have broad substrate specificity and present a 2.1Å crystal structure of IPK from Candidatus methomethylophilus alvus, that identifies molecular factors responsible for broad substrate specificity among IPK. In addition, we will demonstrate the variant IPK generated based on structure-guided engineering to have new activity.

Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor

BackgroundButadiene is a platform chemical used as an industrial feedstock for the manufacture of automobile tires, synthetic resins, latex and engineering plastics. Currently, butadiene is predominantly synthesized as a byproduct of ethylene production from non-renewable petroleum resources. Although the idea of biological synthesis of butadiene from sugars has been discussed in the literature, success for that goal has so far not been reported. As a model system for methanol assimilation, Methylobacterium extorquens AM1 can produce several unique metabolic intermediates for the production of value-added chemicals, including crotonyl-CoA as a potential precursor for butadiene synthesis.ResultsIn this work, we focused on constructing a metabolic pathway to convert crotonyl-CoA into crotyl diphosphate, a direct precursor of butadiene. The engineered pathway consists of three identified enzymes, a hydroxyethylthiazole kinase (THK) from Escherichia coli, an isopentenyl phosphate kinase (IPK) from Methanothermobacter thermautotrophicus and an aldehyde/alcohol dehydrogenase (ADHE2) from Clostridium acetobutylicum. The Km and kcat of THK, IPK and ADHE2 were determined as 8.35 mM and 1.24 s−1, 1.28 mM and 153.14 s−1, and 2.34 mM and 1.15 s−1 towards crotonol, crotyl monophosphate and crotonyl-CoA, respectively. Then, the activity of one of rate-limiting enzymes, THK, was optimized by random mutagenesis coupled with a developed high-throughput screening colorimetric assay. The resulting variant (THKM82V) isolated from over 3000 colonies showed 8.6-fold higher activity than wild-type, which helped increase the titer of crotyl diphosphate to 0.76 mM, corresponding to a 7.6% conversion from crotonol in the one-pot in vitro reaction. Overexpression of native ADHE2, IPK with THKM82V under a strong promoter mxaF in M. extorquens AM1 did not produce crotyl diphosphate from crotonyl-CoA, but the engineered strain did generate 0.60 μg/mL of intracellular crotyl diphosphate from exogenously supplied crotonol at mid-exponential phase.ConclusionsThese results represent the first step in producing a butadiene precursor in recombinant M. extorquens AM1. It not only demonstrates the feasibility of converting crotonol to key intermediates for butadiene biosynthesis, it also suggests future directions for improving catalytic efficiency of aldehyde/alcohol dehydrogenase to produce butadiene precursor from methanol.

Modified mevalonate pathway of the archaeon Aeropyrum pernix proceeds via trans-anhydromevalonate 5-phosphate

The modified mevalonate pathway is believed to be the upstream biosynthetic route for isoprenoids in general archaea. The partially identified pathway has been proposed to explain a mystery surrounding the lack of phosphomevalonate kinase and diphosphomevalonate decarboxylase by the discovery of a conserved enzyme, isopentenyl phosphate kinase. Phosphomevalonate decarboxylase was considered to be the missing link that would fill the vacancy in the pathway between mevalonate 5-phosphate and isopentenyl phosphate. This enzyme was recently discovered from haloarchaea and certain Chroloflexi bacteria, but their enzymes are close homologs of diphosphomevalonate decarboxylase, which are absent in most archaea. In this study, we used comparative genomic analysis to find two enzymes from a hyperthermophilic archaeon, Aeropyrum pernix, that can replace phosphomevalonate decarboxylase. One enzyme, which has been annotated as putative aconitase, catalyzes the dehydration of mevalonate 5-phosphate to form a previously unknown intermediate, trans-anhydromevalonate 5-phosphate. Then, another enzyme belonging to the UbiD-decarboxylase family, which likely requires a UbiX-like partner, converts the intermediate into isopentenyl phosphate. Their activities were confirmed by in vitro assay with recombinant enzymes and were also detected in cell-free extract from A. pernix These data distinguish the modified mevalonate pathway of A. pernix and likely, of the majority of archaea from all known mevalonate pathways, such as the eukaryote-type classical pathway, the haloarchaea-type modified pathway, and another modified pathway recently discovered from Thermoplasma acidophilum.

Contribution of isopentenyl phosphate to plant terpenoid metabolism

Plant genomes encode isopentenyl phosphate kinases (IPKs) that reactivate isopentenyl phosphate (IP) via ATP-dependent phosphorylation, forming the primary metabolite isopentenyl diphosphate (IPP) used generally for isoprenoid/terpenoid biosynthesis. Therefore, the existence of IPKs in plants raises unanswered questions concerning the origin and regulatory roles of IP in plant terpenoid metabolism. Here, we provide genetic and biochemical evidence showing that IP forms during specific dephosphorylation of IPP catalysed by a subset of Nudix superfamily hydrolases. Increasing metabolically available IP by overexpression of a bacterial phosphomevalonate decarboxylase (MPD) in Nicotiana tabacum resulted in significant enhancement in both monoterpene and sesquiterpene production. These results indicate that perturbing IP metabolism results in measurable changes in terpene products derived from both the methylerythritol phosphate (MEP) and mevalonate (MVA) pathways. Moreover, the unpredicted peroxisomal localization of bacterial MPD led us to discover that the step catalysed by phosphomevalonate kinase (PMK) imposes a hidden constraint on flux through the classical MVA pathway. These complementary findings fundamentally alter conventional views of metabolic regulation of terpenoid metabolism in plants and provide new metabolic engineering targets for the production of high-value terpenes in plants.

Reaction Mechanism of Isopentenyl Phosphate Kinase: A QM/MM Study.

Isopentenyl phosphate kinase (IPK) catalyzes the Mg2+-ATP dependent phosphorylation reactions to produce isopentenyl diphosphate, an important precursor in the synthesis of isopentenols. However, the position of the divalent metal ion in the crystal structures of IPK in complex with ATP and its native substrate IP has not been definitively resolved, and as a result ambiguity surrounds the catalytic mechanism of IP, limiting its exploitation as a biofuel and in drug design. Here we report the catalytically competent structure in complex with the metal ion Mg2+ and elucidate the phosphorylation reaction mechanism using molecular dynamic simulations and density functional theory-based quantum mechanics/molecular mechanics calculations (B97d/AMBER99). Comparing the substrate-bound and substrate-free IPK complexes, we observed that substrate binding results in significant conformational change of three residues Lys204, Glu207, and Lys211 located on the αG helix to form a strong salt bridge network with Asp145, which in turn tethers the invariant Ser142 via H-bond interaction. The conformational change shuts the subtrate entrance channel formed between the αG and αE helices. Further, we demonstrate the phosphorylation reaction occurs with a reaction barrier of 17.58 kcal/mol, which is in agreement with the previous experimental kinetic data. We found that a highly conserved Gly8 on a glycine-rich loop, together with Lys14, stabilizes the transition state.

An Adaptation To Life In Acid Through A Novel Mevalonate Pathway.

Extreme acidophiles are capable of growth at pH values near zero. Sustaining life in acidic environments requires extensive adaptations of membranes, proton pumps, and DNA repair mechanisms. Here we describe an adaptation of a core biochemical pathway, the mevalonate pathway, in extreme acidophiles. Two previously known mevalonate pathways involve ATP dependent decarboxylation of either mevalonate 5-phosphate or mevalonate 5-pyrophosphate, in which a single enzyme carries out two essential steps: (1) phosphorylation of the mevalonate moiety at the 3-OH position and (2) subsequent decarboxylation. We now demonstrate that in extreme acidophiles, decarboxylation is carried out by two separate steps: previously identified enzymes generate mevalonate 3,5-bisphosphate and a new decarboxylase we describe here, mevalonate 3,5-bisphosphate decarboxylase, produces isopentenyl phosphate. Why use two enzymes in acidophiles when one enzyme provides both functionalities in all other organisms examined to date? We find that at low pH, the dual function enzyme, mevalonate 5-phosphate decarboxylase is unable to carry out the first phosphorylation step, yet retains its ability to perform decarboxylation. We therefore propose that extreme acidophiles had to replace the dual-purpose enzyme with two specialized enzymes to efficiently produce isoprenoids in extremely acidic environments.