The structural basis of chain length control in Rv1086

In Mycobacterium tuberculosis, two related Z-prenyl diphosphate synthases, E,Z-farnesyl diphosphate synthase (Rv1086) and decaprenyl diphosphate synthase (Rv2361c), work in series to synthesize decaprenyl phosphate (C₅₀) from isopentenyl diphosphate and E-geranyl diphosphate. Decaprenyl phosphate plays a central role in the biosynthesis of essential mycobacterial cell wall components, such as the mycolyl-arabinogalactan-peptidoglycan complex and lipoarabinomannan; thus, its synthesis has attracted considerable interest as a potential therapeutic target. Rv1086 is a unique prenyl diphosphate synthase in that it adds only one isoprene unit to geranyl diphosphate, generating the 15-carbon product (E,Z-farnesyl diphosphate). Rv2361c then adds a further seven isoprene units to E,Z-farnesyl diphosphate in a processive manner to generate the 50-carbon prenyl diphosphate, which is then dephosphorylated to generate a carrier for activated sugars. The molecular basis for chain-length discrimination by Rv1086 during synthesis is unknown. We also report the structure of apo Rv1086 with citronellyl diphosphate bound and with the product mimic E,E-farnesyl diphosphate bound. We report the structures of Rv2361c in the apo form, with isopentenyl diphosphate bound and with a substrate analogue, citronellyl diphosphate. The structures confirm the enzymes are very closely related. Detailed comparison reveals structural differences that account for chain-length control in Rv1086. We have tested this hypothesis and have identified a double mutant of Rv1086 that makes a range of longer lipid chains.     

Product chain-length determination mechanism of Z,E-farnesyl diphosphate synthase

cis-Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic prenyl diphosphates, producing Z,E-mixed prenyl diphosphate. The Mycobacterium tuberculosis Z,E-farnesyl diphosphate synthase Rv1086 catalyzes the condensation of one molecule of IPP with geranyl diphosphate to yield Z,E-farnesyl diphosphate and is classified as a short-chain cis-prenyltransferase. To elucidate the chain-length determination mechanism of the short-chain cis-prenyltransferase, we introduced some substitutive mutations at the characteristic amino acid residues of Rv1086. Among the mutants constructed, L84A showed a dramatic change of catalytic function to synthesize longer prenyl chain products than that of wild type, indicating that Leu84 of Rv1086 plays an important role in product chain-length determination. Mutagenesis at the corresponding residue of a medium-chain cis-prenyltransferase, Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase also resulted in the production of different prenyl chain length from the intrinsic product, suggesting that this position also plays an important role in product chain-length determination for medium-chain cis-prenyltransferases.     

Connected cavity structure enables prenyl elongation across the dimer interface in mutated geranylfarnesyl diphosphate synthase from Methanosarcina mazei

The mechanism for product chain-length determination of geranylfarnesyl diphosphate synthase from the methanogenic archaeon Methanosarcina mazei was investigated by constructing mutants based on structural information. Among the mutants, in which each of the bulky residues that constitute the bottom of the product-accommodating cavity was replaced with alanine, those having mutations on an α-helix existing at the subunit interface yielded longer products. In particular, replacement of isoleucine 112 on the α-helix greatly elongated the product chain-length, probably by connecting the reaction cavities of two subunits across the dimer interface.     

Rv0989c encodes a novel (E)-geranyl diphosphate synthase facilitating decaprenyl diphosphate biosynthesis in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) has a highly complex cell wall, which is required for both bacterial survival and infection. Cell wall biosynthesis is dependent on decaprenyl diphosphate as a glyco-carrier, which is hence an essential metabolite in this pathogen. Previous biochemical studies indicated (E)-geranyl diphosphate (GPP) is required for the synthesis of decaprenyl diphosphate. Here we demonstrate that Rv0989c encodes the “missing” GPP synthase, representing the first such enzyme to be characterized from bacteria, and which presumably is involved in decaprenyl diphosphate biosynthesis in Mtb. Our investigation also has revealed previously unrecognized substrate plasticity of the farnesyl diphosphate synthases from Mtb, resolving previous discrepancies between biochemical and genetic studies of cell wall biosynthesis.     

Cloning and functional characterisation of a cis-muuroladiene synthase from black peppermint (Menthaxpiperita) and direct evidence for a chemotype unable to synthesise farnesene

Using oligonucleotide primers designed to the known gene sequence of an (E)-beta-farnesene (EbetaF) synthase, two cDNA sequences (MxpSS1 and MxpSS2) were cloned from a black peppermint (Menthaxpiperita) plant. MxpSS1 encoded a protein with 96% overall amino acid sequence identity with the EbetaF synthase. Recombinant MxpSS1 produced in Escherichia coli, after removal of an N-terminal thioredoxin fusion, had a K(m) for FPP of 1.91+/-0.1 microM and k(cat) of 0.18 s(-1), and converted farnesyl diphosphate (FPP) into four products, the major two being cis-muurola-3,5-diene (45%) and cis-muurola-4(14),5-diene (43%). This is the first cis-muuroladiene synthase, to be characterised. MxpSS2 encoded a protein with only two amino acids differing from EbetaF synthase. Recombinant MxpSS2 protein showed no activity towards FPP. One of the two mutations, at position 531 (leucine in MxpSS2 and serine in EbetaF synthase) was shown, by structural modelling to occur in the J-K loop, an element of the structure of sesquiterpene synthases known to be important in the reaction mechanism. Reintroduction of the serine at position 531 into MxpSS2 by site-directed mutagenesis restored EbetaF synthase activity (K(m) for FPP 0.98+/-0.12 microM, k(cat) 0.1 s(-1)), demonstrating the crucial role of this residue in the enzyme activity. Analysis, by GC-MS, of the sesquiterpene profile of the plant used for the cloning, revealed that EbetaF was not present, confirming that this particular mint chemotype had lost EbetaF synthase activity due to the observed mutations.     

Geranyl diphosphate synthase from Abies grandis: cDNA isolation,functional expression,and characterization

Geranyl diphosphate synthase catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to generate geranyl diphosphate, the essential precursor of monoterpene biosynthesis. Using geranylgeranyl diphosphate synthase from Taxus canadensis as a hybridization probe, four full length cDNA clones, sharing high sequence identity to each other (>69%) and to the Taxus geranylgeranyl diphosphate synthase (>66%), were isolated from a grand fir (Abies grandis) cDNA library. When expressed in Escherichia coli, three of the recombinant enzymes produced geranyl diphosphate and one produced geranylgeranyl diphosphate as the dominant product when supplied with isopentenyl diphosphate and dimethylallyl diphosphate as cosubstrates. One enzyme (AgGPPS2) was confirmed as a specific geranyl diphosphate synthase, in that it accepted only dimethylallyl diphosphate as the allylic cosubstrate and it produced exclusively geranyl diphosphate as product, with a k(cat) of 1.8s(-1). Gel filtration experiments performed on the recombinant geranyl diphosphate synthases, in which the plastidial targeting sequences had been deleted, revealed that these enzymes are homodimers similar to other short-chain prenyltransferases but different from the heterotetrameric geranyl diphosphate synthase of mint.     

The subcellular localization of periwinkle farnesyl diphosphate synthase provides insight into the role of peroxisome in isoprenoid biosynthesis

Farnesyl diphosphate (FPP) synthase (FPS: EC.2.5.1.1, EC.2.5.1.10) catalyzes the formation of FPP from isopentenyl diphosphate and dimethylallyl diphosphate via two successive condensation reactions. A cDNA designated CrFPS, encoding a protein showing high similarities with trans-type short FPS isoforms, was isolated from the Madagascar periwinkle (Catharanthus roseus). This cDNA was shown to functionally complement the lethal FPS deletion mutant in the yeast Saccharomyces cerevisiae. At the subcellular level, while short FPS isoforms are usually described as cytosolic proteins, we showed, using transient transformations of C. roseus cells with yellow fluorescent protein-fused constructs, that CrFPS is targeted to peroxisomes. This finding is discussed in relation to the subcellular distribution of FPS isoforms in plants and animals and opens new perspectives towards the understanding of isoprenoid biosynthesis.     

Structure of Isoprene Synthase Illuminates the Chemical Mechanism of Teragram Atmospheric Carbon Emission

The X-ray crystal structure of recombinant PcISPS (isoprene synthase from gray poplar hybrid Populus × canescens) has been determined at 2.7 Å resolution, and the structure of its complex with three Mg²⁺ and the unreactive substrate analogue dimethylallyl-S-thiolodiphosphate has been determined at 2.8 Å resolution. Analysis of these structures suggests that the generation of isoprene from substrate dimethylallyl diphosphate occurs via a syn-periplanar elimination mechanism in which the diphosphate-leaving group serves as a general base. This chemical mechanism is responsible for the annual atmospheric emission of 100 Tg of isoprene by terrestrial plant life. Importantly, the PcISPS structure promises to guide future protein engineering studies, potentially leading to hydrocarbon fuels and products that do not rely on traditional petrochemical sources.     

Efficient synthesis of trans-polyisoprene compounds using two thermostable enzymes in an organic-aqueous dual-liquid phase system

The trans-polyisoprene compounds are synthesized by trans-isoprenyl diphosphate synthase (IDS) with consecutive condensation of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP). The in vitro condensation by IDS does not proceed efficiently by hydrophobic interaction between IDS and the hydrocarbon of longer products. In the present study, the enzymatic synthesis of trans-polyisoprenyl diphosphates was attempted in an organic-aqueous dual-liquid phase system with thermostable enzymes obtained from Thermococcus kodakaraensis. The conversion from DMAPP to a longer-chain product was achieved in a dual-liquid phase system, and more than 80% of the products were recovered in the organic phase. When the mutant IDS-Y81S, in which Tyr81 is replaced with Ser, was used in the dual-phase system, productivity was enhanced about four times and the ratio of the longer-chain products was increased. Co-incubation of IPP isomerase from T. kodakaraensis with IDS or IDS-Y81S enabled the direct synthesis of polyisoprenyl diphosphates from IPPs.     

Alkali-Resistant,Alkaline Endo-l,4-β-glucanase Produced by Bacillus sp. PKM-5430

Multiple alignments of primary structures of many kinds of prenyltransferases that participate in the most fundamental prenyl-chain backbone synthesizing process in isoprenoid biosynthesis showed seven conserved regions in the primary structures of (E)-prenyl diphosphate synthases. However, no information has been available about the structures of (Z)-prenyl diphosphate synthases until our recent isolation of the gene for the undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26.

The amino acid sequence of the (Z)-prenyl diphosphate synthase is totally different from those of (E)-prenyl chain elongating enzymes. Protein data base searches for sequences similar to that of the undecaprenyl diphosphate synthase yielded many unknown proteins which have not yet been characterized. Two of the proteins have recently been identified as the undecaprenyl diphosphate synthase of Escherichia coli and the dehydrodolichyl diphosphate synthase of Saccharomyces cerevisiae, indicating that there are three highly conserved regions in the primary structure of (Z)-prenyl chain elongating enzymes.     

Heteromerization of short-chain trans-prenyltransferase controls precursor allocation within a plastidial terpenoid network

Terpenes are the largest and most diverse class of plant specialized metabolites. Sesterterpenes(C25), which are derived from the plastid methylerythritol phosphate pathway,were recently characterized in plants. In Arabidopsis thaliana, four genes encoding geranylfarnesyl diphosphate synthase(GFPPS)(AtGFPPS1 to 4) are responsible for the production of GFPP, which is the common precursor for sesterterpene biosynthesis. However,the interplay between sesterterpenes and other known terpenes remain e...     

A subunit of decaprenyl diphosphate synthase stabilizes octaprenyl diphosphate synthase in Escherichia coli by forming a high-molecular weight complex

The length of the isoprenoid-side chain in ubiquinone, an essential component of the electron transport chain, is defined by poly-prenyl diphosphate synthase, which comprises either homomers (e.g., IspB in Escherichia coli) or heteromers (e.g., decaprenyl diphosphate synthase (Dps1) and D-less polyprenyl diphosphate synthase (Dlp1) in Schizosaccharomyces pombe and in humans). We found that expression of either dlp1 or dps1 recovered the thermo-sensitive growth of an E. coli ispB^R321A mutant and restored IspB activity and production of Coenzyme Q-8. IspB interacted with Dlp1 (or Dps1), forming a high-molecular weight complex that stabilized IspB, leading to full functionality.

Structured summary:

MINT-7385426:Dlp1 (uniprotkb:Q86YH6) and IspB (uniprotkb:P0AD57) physically interact (MI:0915) by blue native page (MI:0276)MINT-7385083, MINT-7385058:IspB (uniprotkb:P0AD57) and IspB (uniprotkb:P0AD57) bind (MI:0407) by blue native page (MI:0276)MINT-7385413:Dlp1 (uniprotkb:O13851) and IspB (uniprotkb:P0AD57) physically interact (MI:0915) by blue native page (MI:0276)MINT-7385024:IspB (uniprotkb:P0AD57) physically interacts (MI:0915) with Dps1 (uniprotkb:O43091) by pull down (MI:0096)MINT-7385041:IspB (uniprotkb:P0AD57) physically interacts (MI:0915) with Dlp1 (uniprotkb:O13851) by pull down (MI:0096)MINT-7385388:IspB (uniprotkb:P0AD57) and Dps1 (uniprotkb:O43091) physically interact (MI:0915) by blue native page (MI:0276)     

Molecular regulation of santalol biosynthesis in Santalum album L.

Santalum album L. commonly known as East-Indian sandal or chandan is a hemiparasitic tree of family santalaceae. Santalol is a bioprospecting molecule present in sandalwood and any effort towards metabolic engineering of this important moiety would require knowledge on gene regulation. Santalol is a sesquiterpene synthesized through mevalonate or non-mevalonate pathways. First step of santalol biosynthesis involves head to tail condensation of isopentenyl pyrophosphate (IPP) with its allylic co-substrate dimethyl allyl pyrophosphate (DMAPP) to produce geranyl pyrophosphate (GPP; C10 — a monoterpene). GPP upon one additional condensation with IPP produces farnesyl pyrophosphate (FPP; C15 — an open chain sesquiterpene). Both the reactions are catalyzed by farnesyl diphosphate synthase (FDS). Santalene synthase (SS), a terpene cyclase catalyzes cyclization of open ring FPP into a mixture of cyclic sesquiterpenes such as α-santalene, epi-β-santalene, β-santalene and exo bergamotene, the main constituents of sandal oil. The objective of the present work was to generate a comprehensive knowledge on the genes involved in santalol production and study their molecular regulation. To achieve this, sequences encoding farnesyl diphosphate synthase and santalene synthase were isolated from sandalwood using suppression subtraction hybridization and 2D gel electrophoresis technology. Functional characterization of both the genes was done through enzyme assays and tissue-specific expression of both the genes was studied. To our knowledge, this is the first report on studies on molecular regulation, and tissue-specific expression of the genes involved in santalol biosynthesis.     

Genomic organization and expression analysis of a farnesyl diphosphate synthase gene (FPPS2) in apples (Malus domestica Borkh.)

A farnesyl diphosphate synthase gene (FPPS2), which contains 11 introns and 12 exons, was isolated from the apple cultivar “White Winter Pearmain”. When it was compared to our previously reported FPPS1, its each intron size was different, its each exon size was the same as that of FPPS1 gene, 30 nucleotide differences were found in its coding sequence. Based on these nucleotide differences, specific primers were designed to perform expression analysis; the results showed that it expressed in both fruit and leaf, its expression level was obviously lower than that of FPPS1 gene in fruit which was stored at 4 °C for 5 weeks. This is the first report concerning two FPPS genes and their expression comparison in apples.     

Inhibition of monoterpene cyclases by inert analogues of geranyl diphosphate and linalyl diphosphate

The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which then cyclizes to the various monoterpene skeletons. X-ray crystal structures of these enzymes complexed with suitable analogues of the substrate and intermediate could provide a clearer view of this universal, but cryptic, step of monoterpenoid cyclase catalysis. Toward this end, the functionally inert analogues 2-fluorogeranyl diphosphate, (±)-2-fluorolinalyl diphosphate, and (3R)- and (3S)-homolinalyl diphosphates (2,6-dimethyl-2-vinyl-5-heptenyl diphosphates) were prepared, and compared to the previously described substrate analogue 3-azageranyl diphosphate (3-aza-2,3-dihydrogeranyl diphosphate) as inhibitors and potential crystallization aids with two representative monoterpenoid cyclases, (-)-limonene synthase and (+)-bornyl diphosphate synthase. Although these enantioselective synthases readily distinguished between (3R)- and (3S)-homolinalyl diphosphates, both of which were more effective inhibitors than was 3-azageranyl diphosphate, the fluorinated analogues proved to be the most potent competitive inhibitors and have recently yielded informative liganded structures with limonene synthase.     

Expression, purification and characterization of recombinant (E)-beta-farnesene synthase from Artemisia annua

A cDNA clone (GenBank Accession No. AY835398) encoding a sesquiterpene synthase, (E)-β-farnesene synthase, has been isolated from Artemisia annua L. It contains a 1746-bp open reading frame coding for 574 amino acids (66.9 kDa) with a calculated pI = 5.03. The deduced amino acid sequence is 30-50% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a single product, β-farnesene, from farnesyl diphosphate. The pH optimum for the recombinant enzyme is around 6.5 and the K_m- and k_cat-values for farnesyl diphosphate, is 2.1 μM and 9.5 × 10⁻³ s⁻¹, respectively resulting in the efficiency 4.5 × 10⁻³ M⁻¹ s⁻¹. The enzyme exhibits substantial activity in the presence of Mg²⁺, Mn²⁺ or Co²⁺ but essentially no activity when Zn²⁺, Ni²⁺ or Cu²⁺ is used as cofactor. The concentration required for maximum activity are estimated to 5 mM, 0.5 mM and <10 μM for Mg²⁺, Co²⁺ or Mn²⁺, respectively. Geranyl diphosphate is not a substrate for the recombinant enzyme.     

Gibberellin biosynthesis in bacteria: Separate ent-copalyl diphosphate and ent-kaurene synthases in Bradyrhizobium japonicum

Gibberellins are ent-kaurene-derived diterpenoid phytohormones produced by plants, fungi, and bacteria. The distinct gibberellin biosynthetic pathways in plants and fungi are known, but not that in bacteria. Plants typically use two diterpene synthases to form ent-kaurene, while fungi use only a single bifunctional diterpene synthase. We demonstrate here that Bradyrhizobium japonicum encodes separate ent-copalyl diphosphate and ent-kaurene synthases. These are found in an operon whose enzymatic composition indicates that gibberellin biosynthesis in bacteria represents a third independently assembled pathway relative to plants and fungi. Nevertheless, sequence comparisons also suggest potential homology between diterpene synthases from bacteria, plants, and fungi.     

Engineered heterologous FPP synthases-mediated Z,E-FPP synthesis in E. coli

Production of Z-type farnesyl diphosphate (FPP) has not been reported in Escherichia coli. Here we present the fusion enzyme (ILRv) of E. coli E,E-FPP synthase (IspA) and Mycobacterium tuberculosis Z,E-FPP synthase (Rv1086), which can produce primarily Z,E-FPP rather than E,E-FPP, the predominant stereoisomer found in most organisms. Z,E-farnesol (FOH) was produced from E. coli harboring the bottom portion of the MVA pathway and the fusion FPP synthase (ILRv) at a titer of 115.6 mg/L in 2 YT medium containing 1% (v/v) glycerol as a carbon source and 5 mM mevalonate. The Z,E-FOH production was improved by 15-fold, compared with 7.7 mg/L obtained from the co-overexpression of separate IspA and Rv1086. The Z,E-FPP was not metabolized in native metabolic pathways of E. coli. It would be of interest to produce Z,E-FPP derived sesquiterpenes from recombinant E. coli due to no loss of Z,E-FPP substrate in endogenous metabolism of the host strain.