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.     

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.     

Arabidopsis thaliana isoprenyl diphosphate synthases produce the C25 intermediate geranylfarnesyl diphosphate

Isoprenyl diphosphate synthases (IDSs) catalyze some of the most basic steps in terpene biosynthesis by producing the prenyl diphosphate precursors of each of the various terpenoid classes. Most plants investigated have distinct enzymes that produce the short‐chain all‐trans (E) prenyl diphosphates geranyl diphosphate (GDP, C₁₀), farnesyl diphosphate (FDP, C₁₅) or geranylgeranyl diphosphate (GGDP, C₂₀). In the genome of Arabidopsis thaliana, 15 trans‐product‐forming IDSs are present. Ten of these have recently been shown to produce GGDP by genetic complementation of a carotenoid pathway engineered into Escherichia coli. When verifying the product pattern of IDSs producing GGDP by a new LC‐MS/MS procedure, we found that five of these IDSs produce geranylfarnesyl diphosphate (GFDP, C₂₅) instead of GGDP as their major product in enzyme assays performed in vitro. Over‐expression of one of the GFDP synthases in A. thaliana confirmed the production of GFDP in vivo. Enzyme assays with A. thaliana protein extracts from roots but not other organs showed formation of GFDP. Furthermore, GFDP itself was detected in root extracts. Subcellular localization studies in leaves indicated that four of the GFDP synthases were targeted to the plastoglobules of the chloroplast and one was targeted to the mitochondria. Sequence comparison and mutational studies showed that the size of the R group of the 5th amino acid residue N‐terminal to the first aspartate‐rich motif is responsible for C₂₅ versus C₂₀ product formation, with smaller R groups (Ala and Ser) resulting in GGDP (C₂₀) as a product and a larger R group (Met) resulting in GFDP (C₂₅).     

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...     

Structural characterization and agonist binding to human α4β2 nicotinic receptors

The Cys-loop receptor super-family of neurotransmitter-gated ion channels mediates fast synaptic transmission throughout the human nervous system. These receptors exhibit widely varying pharmacologies, yet their structural characterization has relied heavily on their homology with the naturally abundant muscle-type Torpedo nicotinic acetylcholine receptor. Here we examine for the first time the structure of a human α4β2 neuronal nicotinic acetylcholine receptor. We show that human α4β2 nicotinic receptors adopt a secondary/tertiary fold similar to that of the Torpedo nicotinic receptor with a large proportion of both α-helix and β-sheet, but exhibit a substantially increased thermal stability. Both receptors bind agonist, but with different patterns of agonist recognition – particularly in the nature of the interactions between aromatic residues and the agonist quaternary amine functional group. By comparing α4β2 and Torpedo receptors, we begin to delineate their structural similarities and differences.     

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.     

Identification of a second catalytically active trans-sialidase in Trypanosoma brucei

The procyclic stage of Trypanosoma brucei is covered by glycosylphosphatidylinositol (GPI)-anchored surface proteins called procyclins. The procyclin GPI anchor contains a side chain of N-acetyllactosamine repeats terminated by sialic acids. Sialic acid modification is mediated by trans-sialidases expressed on the parasite’s cell surface. Previous studies suggested the presence of more than one active trans-sialidases, but only one has so far been reported. Here we cloned and examined enzyme activities of four additional trans-sialidase homologs, and show that one of them, Tb927.8.7350, encodes another active trans-sialidase, designated as TbSA C2. In an in vitro assay, TbSA C2 utilized α2-3 sialyllactose as a donor, and produced an α2-3-sialylated product, suggesting that it is an α2-3 trans-sialidase. We suggest that TbSA C2 plays a role in the sialic acid modification of the trypanosome cell surface.     

Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: Purification and characterization of the inhibitory complex

Saccharomyces cerevisiae antizyme (AZ) resembles mammalian AZ in its mode of synthesis by translational frameshifting and its ability to inhibit and facilitate the degradation of ornithine decarboxylase (ODC). Despite many studies on the interaction of AZ and ODC, the ODC:AZ complex has not been purified from any source and thus clear information about the stoichiometry of the complex is still lacking. In this study we have studied the yeast antizyme protein and the ODC:AZ complex. The far UV CD spectrum of the full-length antizyme shows that the yeast protein consists of 51% β-sheet, 19% α-helix, and 24% coils. Surface plasmon resonance analyses show that the association constant (K_A) between yeast AZ and yeast ODC is 6 × 10⁷ (M⁻¹). Using purified His-tagged AZ as a binding partner, we have purified the ODC:AZ inhibitory complex. The isolated complex has no ODC activity. The molecular weight of the complex is 90 kDa, which indicates a one to one stoichiometric binding of AZ and ODC in vitro. Comparison of the circular dichroism (CD) spectra of the two individual proteins and of the ODC:AZ complex shows a change in the secondary structure in the complex.     

Differential interactions of cerebellin precursor protein (Cbln) subtypes and neurexin variants for synapse formation of cortical neurons

The polyisoprenoid alcohols (dolichols and polyprenols) are found in all living organism, from bacteria to mammals. In animal and yeast cells polyisoprenoids are derived from the cytoplasmic mevalonate (MVA) pathway while in plants two biosynthetic pathways, the MVA and the plastidial methylerythritol phosphate (MEP) pathway provide precursors for polyisoprenoid biosynthesis. The key enzymes of polyisoprenoid synthesis are cis-prenyltransferases (CTPs), responsible for construction of the long hydrocarbon skeleton. CPTs elongate a short all-trans precursor, oligoprenyl diphosphate, by sequential addition of the desired number of isopentenyl diphosphate molecules which results in formation of a stretch of cis units. Several genes encoding CPT have been cloned from bacteria, plants and mammals. Interestingly, in Arabidopsis, the tissue-specific expression of ten putative cis-prenyltransferases was observed. In contrast to polyisoprenoid phosphates serving as cofactors in the biosynthesis of glycoproteins, glucosyl phosphatidyl inositol (GPI) anchor or bacterial peptidoglycan, the biological importance of polyprenols and dolichols still remains a question of debate besides their function of reservoir of substrates for kinase. These extremely hydrophobic superlipids are postulated to be involved in intracellular traffic of proteins and in cellular defense against adverse environmental conditions. Recent publications show a direct link between the dolichol biosynthetic pathway and congenital disorders of glycosylation (CDG). These discoveries highlighting the cellular significance of polyisoprenoids simultaneously establish the background for future pharmacological interventions. Our mini-review summarizes the results of recent studies on polyisoprenoids.     

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.     

An intron-free methyl jasmonate inducible geranylgeranyl diphosphate synthase gene from Taxus media and its functional identification in yeast

Geranylgeranyl diphosphate synthase (GGPPS) [EC 2.5.1.29] catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes and, in particular, Taxol, one of the most potent antitumor drugs. In order to investigate the role of GGPP synthase in Taxol biosynthesis, we cloned, characterized, and functionally expressed the GGPPS gene from Taxus media. Using the genome walking strategy, a 3743-bp genomic sequence of T. media was isolated which contained a 1182-bp open reading frame (ORF) encoding a 393-amino acid polypeptide that showed a close similarity to other plant GGPPSs. Subsequently, the full-length cDNA of the GGPPS gene of T. media (designated TmGGPPS) was amplified by RACE. Bioinformatic analysis showed that TmGGPPS was an intron-free gene, and its deduced polypeptide contained all five conserved domains and functional aspartate-rich motifs of the prenyltransferases. By constructing the phylogenetic tree of plant GGPPSs, it was found that plant-derived GGPPSs could be divided into two classes, those of angiosperms and gymnosperms, which might have evolved in parallel from the same ancestor. To our knowledge, this was the first report that the geranylgeranyl diphosphate synthase genes were free of introns and evolved in parallel in both angiosperms and gymnosperms. The coding sequence of TmGGPPS was expressed through functional complementation in a yeast mutant lacking GGPPS activity (SFNY368), and the transgenic yeast was shown to have this activity. This was also the first time SFNY368 was used to identify the function of plant-derived GGPPSs. Furthermore, investigation of the effect of methyl jasmonate (MeJA) on the expression of TmGGPPS showed that MeJA-treated T. media cultured cells had much higher expression of TmGGPPS than untreated cells.From Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 14–20.Original English Text Copyright © 2005 by Zhihua Liao, Yifu Gong, Guoyin Kai, Kaijing Zuo, Min Chen, Qiumin Tan, Yamin Wei, Liang Guo, Feng Tan, Xiaofen Sun, Kexuan Tang.This article was submitted by the authors in English.     

Knockout of a starch synthase gene <Emphasis Type="Italic">OsSSIIIa</Emphasis>/<Emphasis Type="Italic">Flo5</Emphasis> causes white-core floury endosperm in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

To elucidate the role of SSIIIa during starch synthesis in rice (Oryza sativa L.) endosperm, we characterized null mutants of this gene, generated by T-DNA insertions. Scanning electron microscope (SEM) analysis revealed that the starch granules in these mutants are smaller and rounder compared with the wild type controls, and that the mutant endosperm is characterized by a loosely packed central portion exhibiting a floury-like phenotype. Hence, the OsSSIIIa (Oryza sativa SSIIIa) mutations are referred to as white-core floury endosperm 5-1 (flo5-1) and flo5-2. Based upon their X-ray diffraction patterns, the crystallinity of the starch in the flo5 mutant endosperm is decreased compared with wild type. Through determination of the chain-length distribution of the mutant endosperm starch, we found that flo5-1 and flo5-2 mutants have reduced the content of long chains with degree of polymerization (DP) 30 or greater compared with the controls. This suggests that OsSSIIIa/Flo5 plays an important role in generating relatively long chains in rice endosperm. In addition, DP 6 to 8 and DP 16 to 20 appeared to be reduced in endosperm starch of flo5-1 and flo5-2, whereas DP 9 to 15 and DP 22 to 29 were increased in these mutants. By the use of differential scanning calorimetry (DSC), the gelatinization temperatures of endosperm starch were found to be 1–5°C lower than those of the control. We propose a distinct role for OsSSIIIa/Flo5 and the coordinated action of other SS isoforms during starch synthesis in the seed endosperm of rice.     

A New Form of Structural Peptidoglycan in Selenomonas ruminantium: Existence of Polyamine in Peptidoglycan

The chemical constituents of calluses of Thujopsis dolabrata (Hiba), Chamaecyparis obtusa (Hinoki), Chamaecyparis pisifera (Sawara), and Platycladus orientalis (Konotegashiwa), all of which belong to Cupressaceae, were examined by GC analysis. The main components of these calluses were diterpenoids of an abietane-type and sitosterol in common, while the chemical constituents of these parent plants were different from each other.     

Configuration of polyisoprenoids affects the permeability and thermotropic properties of phospholipid/polyisoprenoid model membranes

The influence of α-cis- and α-trans-polyprenols on the structure and properties of model membranes was analyzed. The interaction of Ficaprenol-12 (α-cis-Prenol-12, α-Z-Prenol-12) and Alloprenol-12 (α-trans-Prenol-12, α-E-Prenol-12) with model membranes was compared using high performance liquid chromatography (HPLC), differential scanning calorimetry (DSC) and fluorescent methods. l-α-Phosphatidylcholine from egg yolk (EYPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as the main lipid components of unilamellar (SUVs) and multilamellar (MLVs) vesicles were used. The two-step extraction procedure (n-pentane and hexane, respectively) allowed to separately analyze the fractions of polyprenol as non-incorporated (Prenol_NonInc) and incorporated (Prenol_Inc) into liposomes. Consequently, distribution coefficients, P′, describing the equilibrium of prenol content between phospholipid (EYPC) membrane and the aqueous phase gave different log P′ for α-cis- and α-trans-Prenol-12, indicating that the configuration of the α-terminal residue significantly alters the hydrophobicity of the polyisoprenoid molecule and consequently the affinity of polyprenols for EYPC membrane. In fluorescence experiments α-trans-Pren-12 increased up to 1.7-fold the permeability of EYPC bilayer for glucose while the effect of α-cis-Pren-12 was almost negligible. Considerable changes of thermotropic behavior of DPPC membranes in the presence of both prenol isomers were observed. α-trans-Pren-12 completely abolished the pretransition while in the case of α-cis-Pren-12 it was noticeably reduced. Furthermore, for both prenol isomers, the temperature of the main phase transition (T_m) was shifted by about 1 °C to lower values and the height of the peak was significantly reduced. The DSC analysis profiles also showed a new peak at 38.7 °C, which may suggest the concomitant presence of more that one phase within the membrane.Results of these experiments and the concomitant occurrence of alloprenols and ficaprenols in plant tissues suggest that cis/trans isomerization of the α-residue of polyisoprenoid molecule might comprise a putative mechanism responsible for modulation of the permeability of cellular membranes.     

A gene cluster involved in nogalamycin biosynthesis fromStreptomyces nogalater: sequence analysis and complementation of early-block mutations in the anthracycline pathway

We have analyzed an anthracycline biosynthesis gene cluster fromStreptomyces nogalater. Based on sequence analysis, a contiguous region of 11 kb is deduced to include genes for the early steps in anthracycline biosynthesis, a regulatory gene (snoA) promoting the expression of the biosynthetic genes, and at least one gene whose product might have a role in modification of the glycoside moiety. The three ORFs encoding a minimal polyketide synthase (PKS) are separated from the regulatory gene (snoA) by a comparatively AT-rich region (GC content 60%). Subfragments of the DNA region were transferred toStreptomyces galilaeus mutants blocked in aclacinomycin biosynthesis, and to a regulatory mutant ofS. nogalater. TheS. galilaeus mutants carrying theS. nogalater minimal PKS genes produced auramycinone glycosides, demonstrating replacement of the starter unit for polyketide biosynthesis. The product ofsnoA seems to be needed for expression of at least the genes for the minimal PKS.     

The role of tropomyosin domains in cooperative activation of the actin-myosin interaction

To establish α-tropomyosin (Tm)'s structure–function relationships in cooperative regulation of muscle contraction, thin filaments were reconstituted with a variety of Tm mutants (Δ2Tm, Δ3Tm, Δ6Tm, P2sTm, P3sTm, P2P3sTm, P1P5Tm, and wtTm), and force and sliding velocity of the thin filament were studied using an in vitro motility assay. In the case of deletion mutants, Δ indicates which of the quasi-equivalent repeats in Tm was deleted. In the case of period (P) mutants, an Ala cluster was introduced into the indicated period to strengthen the Tm–actin interaction. In P1P5Tm, the N-terminal half of period 5 was substituted with that of period 1 to test the quasi-equivalence of these two Tm periods. The reconstitution included bovine cardiac troponin. Deletion studies revealed that period 3 is important for the positive cooperative effect of Tm on actin filament regulation and that period 2 also contributes to this effect at low ionic strength, but to a lesser degree. Furthermore, Tm with one extra Ala cluster at period 2 (P2s) or period 3 (P3s) did not increase force or velocity, whereas Tm with two extra Ala clusters (P2P3s) increased both force and velocity, demonstrating interaction between these periods. Most mutants did not move in the absence of Ca²⁺. Notable exceptions were Δ6Tm and P1P5Tm, which moved near at the full velocity, but with reduced force, which indicate impaired relaxation. These results are consistent with the mechanism that the Tm–actin interaction cooperatively affects actin to result in generation of greater force and velocity.     

Substrate specificities of farnesyl diphosphate synthases from Bacillus stearothermophilus and porcine liver with cyclic substrate homologs

We investigated the substrate specificity of farnesyl diphosphate (FPP) synthase derived from Bacillus stearothermophilus and porcine liver by examining the reactivities of two cyclic substrate homologs, cyclohexylideneethyl diphosphate and cyclohexenylethyl diphosphate.Reaction of geranyl diphosphate with 2-cyclohexenylethyl diphosphate using bacterial or porcine liver FPP synthase produced (S)-geranylcyclohexylideneethyl diphosphate, with relative yields of 13.6% for the bacterial enzyme and 42.2% for the porcine liver enzyme. Reaction of cyclohexylideneethyl diphosphate with isopentenyl diphosphate produced 10-cyclohexyliden-3,7-dimethyldeca-2,6-dien-1-ol as a double condensation product, with relative yields of 23.1% (bacterial enzyme) and 3.0% (porcine liver enzyme). Reaction of cyclohexylideneethyl diphosphate with 2-cyclohexenylethyl diphosphate using bacterial enzyme produced (cyclohexylideneethyl)-cyclohexylideneethyl diphosphate (0.8% yield).     

Computer-aided subsite mapping of α-amylases

Subsite mapping is a crucial procedure in the characterization of α-amylases (EC 3.2.1.1), which are extensively used in starch-based industries and in diagnosis of pancreatic and salivary glands disorders. A computer-aided method has been developed for subsite mapping of α-amylases, which substitutes the difficult, expensive, and time-consuming experimental determination of action patterns to crystal structures based energy calculations. Interaction energies between enzymes and carbohydrate substrates were calculated after short energy minimization by a molecular mechanics program. A training set of wild type and mutant amylases with known experimental action patterns of 13 enzymes of wide range of origin was used to set up the procedure. Calculations for training set resulted in good correlation in case of subsite binding energies (r² = 0.827–0.929) and bond cleavage frequencies (r² = 0.727–0.835). A set of eight novel barley amylase 1 mutants was used to test our model. Subsite binding energies were predicted with r² = 0.502 correlation coefficient, while bond cleavage frequency prediction resulted in r² = 0.538. Our computer-aided procedure may supplement the experimental subsite mapping methods to predict and understand characteristic features of α-amylases.     

Secretion of N-terminal domain of α-dystroglycan in cerebrospinal fluid

α-Dystroglycan (α-DG) plays crucial roles in maintaining the stability of cells. We demonstrated previously that the N-terminal domain of α-DG (α-DG-N) is secreted by cultured cells into the culture medium. In the present study, to clarify its function in vivo, we generated a monoclonal antibody against α-DG-N and investigated the secretion of α-DG-N in human cerebrospinal fluid (CSF). Interestingly, we found that a considerable amount of α-DG-N was present in CSF. α-DG-N in CSF was a sialylated glycoprotein with both N- and O-linked glycan. These observations suggest that secreted α-DG-N may be transported via CSF and have yet unidentified effects on the nervous system.