cis-Prenyltransferase AtCPT6 produces a family of very short-chain polyisoprenoids in planta

cis-Prenyltransferases (CPTs) comprise numerous enzymes synthesizing isoprenoid hydrocarbon skeleton with isoprenoid units in the cis (Z) configuration. The chain-length specificity of a particular plant CPT is in most cases unknown despite the composition of the accumulated isoprenoids in the tissue of interest being well established. In this report AtCPT6, one of the nine Arabidopsis thaliana CPTs, is shown to catalyze the synthesis of a family of very short-chain polyisoprenoid alcohols of six, seven, and eight isoprenoid units, those of seven units dominating. The product specificity of AtCPT6 was established in vivo following its expression in the heterologous system of the yeast Saccharomyces cerevisiae and was confirmed by the absence of specific products in AtCPT6 T-DNA insertion mutants and their overaccumulation in AtCPT6-overexpressing plants. These observations are additionally validated in silico using an AtCPT6 model obtained by homology modeling. AtCPT6 only partially complements the function of the yeast homologue of CPT-Rer2 since it restores the growth but not protein glycosylation in rer2Δ yeast. This is the first in planta characterization of specific products of a plant CPT producing polyisoprenoids. Their distribution suggests that a joint activity of several CPTs is required to produce the complex mixture of polyisoprenoid alcohols found in Arabidopsis roots.     

Polyprenols in hairy roots of Coluria geoides

Long-chain polyisoprenoid alcohols built from several up to more than 100 isoprenoid units are common constituents of all living organisms. They were found mostly in plants, bacteria, yeasts and mammalian cells. In vitro hairy root culture of Coluria geoides was obtained from plants transformed with Agrobacterium rhizogenes. Growth was optimal at 0.75% (w/v) glucose and at 22 degrees C. Dry samples of roots were extracted and lipid content was analysed by HPLC. According to our estimation, polyprenols are accumulated in roots of C. geoides cultivated in vitro as a mixture of several prenologues with the dominating prenol composed of 16 isoprenoid units. The content of polyprenols in tissue was approx. 300 microg/g of dry weight.     

Dolichol: function, metabolism, and accumulation in human tissues.

Dolichol, a homologous series of alpha-saturated polyisoprenoid alcohols containing 14-24 isoprene units, was first isolated and characterized about 30 years ago. The phosphorylated form, dolichyl phosphate, is required for the biosynthesis of biologically important N-linked glycoproteins. Dolichol itself is synthesized by a common isoprenoid pathway from acetate and synthesis can be inhibited by some of the factors that inhibit cholesterol biosynthesis. It is metabolized very slowly and accumulates in tissues during aging and in certain lipid storage diseases. Dolichyl phosphate and cholesterol also accumulate in tissues during aging, but to a lesser extent than dolichol. Although dolichol and cholesterol have important metabolic functions, their accumulation in tissues can have deleterious effects.     

Distribution, metabolism and function of dolichol and polyprenols

Polyisoprenoid alcohols consisting of 9 or more isoprene units are present in all living cells. They can be fully unsaturated (polyprenols) or alpha-saturated (dolichol). Dolichol forms may have additional saturation at or near the omega-end. Some species contain ony dolichol or only polyprenols while others have nearly equal amounts of both types. Some polyisoprenoid alcohols consist entirely of trans isoprene units but most, including dolichol, contain both trans and cis units. Considerable advances in lipid methodology have occurred since the first review of polyisoprenoid alcohols by Hemming in 1974. For example, direct analysis of both dolichol and Dol-P by HPLC has replaced earlier methods which were often both insensitive and inaccurate. The availability of radiolabeled dolichol and polyprenols has facilitated studies concerning the metabolism and distribution of these compounds. Those studies suggest that only a small portion of the dolichol present in cells is likely to be involved in glycosylation. Polyisoprenoid alcohols are usually present at a family of homologues where each differs in size by one isoprene unit. Little or no size related specificity has been observed for any reaction involving dolichol or polyisoprenol intermediates. The overall length of polyisoprenoid alcohols may, however, affect the manner in which these compounds influence the physical and biochemical properties of membranes. Studies on the biosynthetic pathway leading from cis, trans Pol-PP by phosphatase action. The formation of the dolichol backbone from a polyprenol requires the action of an additional enzyme, an alpha-saturase. This enzyme does not always act at the level of a single common substrate, since Pol-PP, Pol-P, and polyprenol all appear to be utilized as substrates. The major product of the de novo pathway differs among different species. Dol-P would appear to be the most energy efficient end-product since it can participate directly in glycoprotein formation. Most often, however, Dol-P is not the major product of metabolic labeling experiments. In some cases, dolichol is formed so that rephosphorylation is required to provide Dol-P for participation in glycoprotein formation. The kinase responsible for this phosphorylation appears to bypass the considerable stores of dolichol present in tissues (i.e. sea urchin eggs) in favor of dolichol derived directly from de novo synthesis. Although HMGR is a major regulatory component of the pathway leading to polyisoprenoid alcohols and cholesterol, control is most often not co-ordinated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Long chain polyisoprenoid alcohols in leaves of Capparis species.

Leaves of twelve species of the genus Capparis were examined for the presence of long chain polyisoprenoid alcohols. In a number of species the accumulation of polyisoprenoid alcohols was up to about 0.3% of dry weight of tissue. In all studied species polyisoprenoid alcohols composed of 12, 13, 14 or 15 isoprene residues formed the main polyprenol family. In the majority of the plants studied lower quantities of an additional polyprenol family were present, in which prenologues composed of 19, 20 or 21 isoprene units were dominating. In one species--Capparis coriacea also the presence of dolichol-like polyprenols with a hydrogenated OH-terminal isoprene unit was documented.     

A gas chromatographic-mass spectrometric analysis of the esterifying alcohols of pumpkin seed protochlorophylls

The esterifying alcohols of protochlorophyll a and 4-vinyl-(4-desethyl)-protochlorophyll a (purified as the respective pheophytins) from pumpkin seeds were examined by gas chromatography-mass spectrometry. The results of the analysis suggested that pumpkin seed protochlorophyll a is esterified with all possible C₂₀ isoprenoid alcohols between and including geranylgeraniol and phytol, phytol comprising 90% or more of the mixture of esterifying alcohols, and that the 4-vinyl-(4-desethyl)-protochlorophyll a is esterified with farnesol and all possible C₂₀ isoprenoid alcohols between and including geranylgeranoid and phytanol, phytol comprising 50% or more of the mixture of esterifying alcohols. The 4-vinyl-(4-desethyl)-protochlorophyll a from a sample of older mature pumpkin seeds was found to be richer in esterifying alcohols corresponding to isoprenoid precursors of phytol then was the 4-vinyl-(4-desethyl)-protochlorophyll a from a sample of younger mature seeds. Other isoprenoid alcohols may have been present in very minor quantities in the mixtures of esterifying alcohols from the pumpkin seed protochlorophylls but were not looked for in this study. These results are discussed in terms of a biosynthetic accumulation of 4-vinyl-(4-desethyl)-protochlorophyll a in pumpkin inner seed-coat tissue.     

Crystal structure of a novel polyisoprenoid-binding protein from Thermus thermophilus HB8

The isoprenoid quinones exist widely among prokaryotes and eukaryotes. They play essential roles in respiratory electron transport and in controlling oxidative stress and gene regulation. In the isoprenoid quinone biosynthetic pathway, polyprenyl pyrophosphates are used as isoprenoid side-chain precursors. Here we report the crystal structure of a novel polyprenyl pyrophosphate binding protein, TT1927b, from Thermus thermophilus HB8, complexed with its ligand. This protein belongs to the YceI-like family in the Pfam database, and its sequence homologs are present in a broad range of bacteria and archaea. The structure consists of an extended, eight-stranded, antiparallel beta-barrel. In the hydrophobic pore of the barrel, the protein binds the polyisoprenoid chain by hydrophobic interactions. Its overall structure resembles the lipocalin fold, but there is no sequence homology between TT1927b and the lipocalin family of proteins.     

Role of polyisoprenoids in tobacco resistance against biotic stresses

Infection with avirulent pathogens, tobacco mosaic virus (TMV) or Pseudomonas syringae pv. tabaci induced accumulation of polyisoprenoid alcohols, solanesol and a family of polyprenols [from polyprenol composed of 14 isoprene units (Pren-14) to -18, with Pren-16 dominating] in the leaves of resistant tobacco plants Nicotiana tabacum cv. Samsun NN. Upon TMV infection, solanesol content was increased seven- and eight-fold in the inoculated and upper leaves, respectively, while polyprenol content was increased 2.5- and 2-fold in the inoculated and upper leaves, respectively, on the seventh day post-infection. Accumulation of polyisoprenoid alcohols was also stimulated by exogenously applied hydrogen peroxide but not by exogenous salicylic acid (SA). On the contrary, neither inoculation of the leaves of susceptible tobacco plants nor wounding of tobacco leaves caused an increase in polyisoprenoid content. Taken together, these results indicate that polyisoprenoid alcohols might be involved in plant resistance against pathogens. A putative role of accumulated polyisoprenoids in plant response to pathogen attack is discussed. Similarly, the content of plastoquinone (PQ) was increased two-fold in TMV-inoculated and upper leaves of resistant plants. Accumulation of PQ was also stimulated by hydrogen peroxide, bacteria ( P.  syringae ) and SA. The role of PQ in antioxidant defense in cellular membranous compartments is discussed in the context of the enzymatic antioxidant machinery activated in tobacco leaves subjected to viral infection. Elevated activity of several antioxidant enzymes (ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and superoxide dismutase, especially the CuZn superoxide dismutase isoform) and high, but transient elevation of catalase was found in inoculated leaves of resistant tobacco plants but not in susceptible plants.     

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.     

Ubiquinone. Biosynthesis of quinone ring and its isoprenoid side chain. Intracellular localization

Ubiquinone, known as coenzyme Q, was shown to be the part of the metabolic pathways by Crane et al. in 1957. Its function as a component of the mitochondrial respiratory chain is well established. However, ubiquinone has recently attracted increasing attention with regard to its function, in the reduced form, as an antioxidant. In ubiquinone synthesis the para-hydroxybenzoate ring (which is the derivative of tyrosine or phenylalanine) is condensed with a hydrophobic polyisoprenoid side chain, whose length varies from 6 to 10 isoprene units depending on the organism. para-Hydroxybenzoate (PHB) polyprenyltransferase that catalyzes the condensation of PHB with polyprenyl diphosphate has a broad substrate specificity. Most of the genes encoding (all-E)-prenyltransferases which synthesize polyisoprenoid chains, have been cloned. Their structure is either homo- or heterodimeric. Genes that encode prenyltransferases catalysing the transfer of the isoprenoid chain to para-hydroxybenzoate were also cloned in bacteria and yeast. To form ubiquinone, prenylated PHB undergoes several modifications such as hydroxylations, O-methylations, methylations and decarboxylation. In eukaryotes ubiquinones were found in the inner mitochondrial membrane and in other membranes such as the endoplasmic reticulum, Golgi vesicles, lysosomes and peroxisomes. Still, the subcellular site of their biosynthesis remains unclear. Considering the diversity of functions of ubiquinones, and their multistep biosynthesis, identification of factors regulating their cellular level remains an elusive task.     

Kinetic studies of AKR1B10, human aldose reductase-like protein: Endogenous substrates and inhibition by steroids

A human member of the aldo-keto reductase (AKR) superfamily, AKR1B10, was identified as a biomarker of lung cancer, exhibiting high sequence identity with human aldose reductase (AKR1B1). Using recombinant AKR1B10 and AKR1B1, we compared their substrate specificity for biogenic compounds and inhibition by endogenous compounds and found the following unique features of AKR1B10. AKR1B10 efficiently reduced long-chain aliphatic aldehydes including farnesal and geranylgeranial, which are generated from degradation of prenylated proteins and metabolism of farnesol and geranylgeraniol derived from the mevalonate pathway. The enzyme oxidized aliphatic and aromatic alcohols including 20α-hydroxysteroids. In addition, AKR1B10 was inhibited by steroid hormones, bile acids and their metabolites, showing IC₅₀ values of 0.03-25 μM. Kinetic analyses of the alcohol oxidation and inhibition by the steroids and tolrestat, together with the docked model of AKR1B10-inhibitor complex, suggest that the inhibitory steroids and tolrestat bind to overlapping sites within the active site of the enzyme-coenzyme complex. Thus, we propose a novel role of AKR1B10 in controlling isoprenoid homeostasis that is important in cholesterol synthesis and cell proliferation through salvaging isoprenoid alcohols, as well as its metabolic regulation by endogenous steroids.     

Metabolic transformation of mevalonic Acid by an enzyme system from peas

En enzyme system has been found in peas which converts mevalonic acid to isoprenoid compounds. Among the intermediates in such conversion are mevalonic acid-5-phosphate and pyrophosphate, isopentenyl pyrophosphate and dimethylallylpyrophosphate. Among the products formed by the system are the pyrophosphates of geraniol, farnesol, nerolidol and higher isoprenoid alcohols.     

Chain elongation in the isoprenoid biosynthetic pathway

Isoprenyl diphosphate synthases catalyze addition of allylic diphosphate primers to the isoprene unit in isopentenyl diphosphate to produce polyisoprenoid diphosphates with well defined chain lengths. Phylogenetic correlations suggest that the synthases which catalyze formation of isoprenoid diphosphates with (E) double bonds have evolved from a common ancestor. X-ray crystallographic studies of farnesyl diphosphate synthase in conjunction with site-directed mutagenesis have provided important new information about the residues involved in binding and catalysis and the source of chain length selectivity for the enzymes that catalyze chain elongation.     

Metabolic engineering for higher alcohol production

Engineering microbial hosts for the production of higher alcohols looks to combine the benefits of renewable biological production with the useful chemical properties of larger alcohols. In this review we outline the array of metabolic engineering strategies employed for the efficient diversion of carbon flux from native biosynthetic pathways to the overproduction of a target alcohol. Strategies for pathway design from amino acid biosynthesis through 2-keto acids, from isoprenoid biosynthesis through pyrophosphate intermediates, from fatty acid biosynthesis and degradation by tailoring chain length specificity, and the use and expansion of natural solvent production pathways will be covered.     

Fatty aldehyde and fatty alcohol metabolism: Review and importance for epidermal structure and function

Normal fatty aldehyde and alcohol metabolism is essential for epidermal differentiation and function. Long-chain aldehydes are produced by catabolism of several lipids including fatty alcohols, sphingolipids, ether glycerolipids, isoprenoid alcohols and certain aliphatic lipids that undergo α- or ω-oxidation. The fatty aldehyde generated by these pathways is chiefly metabolized to fatty acid by fatty aldehyde dehydrogenase (FALDH, alternately known as ALDH3A2), which also functions to oxidize fatty alcohols as a component of the fatty alcohol:NAD oxidoreductase (FAO) enzyme complex. Genetic deficiency of FALDH/FAO in patients with Sjögren–Larsson syndrome (SLS) results in accumulation of fatty aldehydes, fatty alcohols and related lipids (ether glycerolipids, wax esters) in cultured keratinocytes. These biochemical changes are associated with abnormalities in formation of lamellar bodies in the stratum granulosum and impaired delivery of their precursor membranes to the stratum corneum (SC). The defective extracellular SC membranes are responsible for a leaky epidermal water barrier and ichthyosis. Although lamellar bodies appear to be the pathogenic target for abnormal fatty aldehyde/alcohol metabolism in SLS, the precise biochemical mechanisms are yet to be elucidated. Nevertheless, studies in SLS highlight the critical importance of FALDH and normal fatty aldehyde/alcohol metabolism for epidermal function. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Biosynthesis of Organic Compounds Emitted by Plants

Abstract: Trees produce a wide spectrum of volatile organic compounds (VOCs) including isoprene and monoterpenes, as well as oxygenated compounds like aldehydes, alcohols and carboxylic acids. In recent years, much progress has been made regarding the elucidation of metabolic pathways leading to the production of these compounds. This is particularly true for the biosynthesis of the isoprenoid precursors, isopentenyl diphos-phate (lOP) and dimethyl allyl diphosphate (DMADP). In addition to the classical mevalonate pathway which leads to the biosynthesis of these compounds, recent studies indicate the presence of a non-mevalonate pathway originating from pyru-vate and glyceraldehyde-3-phosphate (GAP), also leading to isoprenoid precursors. This new 1-deoxy-D-xylulose-5-phos-phate (DOXP) pathway is probably responsible for the formation of all plastid-derived isoprenoid compounds in plants, including carotenoids, plastochinones, the prenyl side chains of chlorophyll, as well as monoterpenes and diterpenes. Because all plas-tidic isoprenoids studied so far are formed via this new pathway, it is assumed that isoprene synthesized in the chloroplasts is also produced via this metabolic route. Among the oxygenated hydrocarbons which are emitted by the leaves of trees, C-1 and C-2 aldehydes, alcohols and carboxylic acids are of great importance. C-i compounds are synthesized during many growth and developmental processes such as seed maturation, cell expansion, cell wall degradation, leaf abscission and senescence of plant tissues. The production of C-2 compounds, however, seems mainly to be associated with changing environmental conditions, particularly during stress. Acetaldehyde, for example, is produced in the leaves of trees if the roots are exposed to anaerobic conditions which in nature may be caused by flooding. As a consequence of anaerobiosis, roots produce ethanol through alcoholic fermentation. Ethanol is loaded into the xy-lem, transported to the leaves and oxidized there under aerobic conditions, thereby releasing acetaldehyde and acetic acid.     

Engineering metabolic systems for production of advanced fuels

The depleting petroleum storage and increasing environmental deterioration are threatening the sustainable development of human societies. As such, biofuels and chemical feedstocks generated from renewable sources are becoming increasingly important. Although previous efforts led to great success in bio-ethanol production, higher alcohols, fatty acid derivatives including biodiesels, alkanes, and alkenes offer additional advantages because of their compatibility with existing infrastructure. In addition, some of these compounds are useful chemical feedstocks. Since native organisms do not naturally produce these compounds in high quantities, metabolic engineering becomes essential in constructing producing organisms. In this article, we briefly review the four major metabolic systems, the coenzyme-A mediated pathways, the keto acid pathways, the fatty acid pathway, and the isoprenoid pathways, that allow production of these fuel-grade chemicals.     

Mono- and dialkyl isoprenoid bisphosphonates as geranylgeranyl diphosphate synthase inhibitors

Nitrogenous bisphosphonates are used clinically to reduce bone resorption associated with osteoporosis or metastatic bone disease, and are recognized as inhibitors of farnesyl diphosphate synthase. Inhibition of this enzyme decreases cellular levels of both farnesyl diphosphate and geranylgeranyl diphosphate which results in a variety of downstream biological effects including inhibition of protein geranylgeranylation. Our lab recently has prepared several isoprenoid bisphosphonates that inhibit protein geranylgeranylation and showed that one selectively inhibits geranylgeranyl diphosphate synthase. This results in depletion of intracellular geranylgeranyl diphosphate and impacts protein geranylgeranylation but does not affect protein farnesylation. To clarify the structural features of isoprenoid bisphosphonates that account for their geranylgeranyl diphosphate synthase inhibition, we have prepared a new group of isoprenoid bisphosphonates. The complete set of compounds has been tested for in vitro inhibition of human recombinant geranylgeranyl diphosphate synthase and cellular inhibition of protein geranylgeranylation. These results show some surprising relationships between in vitro and cellular activity, and will guide development of clinical agents directed at geranylgeranyl diphosphate synthase.     

Structure and synthesis of polyisoprenoids used in N-glycosylation across the three domains of life

N-linked protein glycosylation was originally thought to be specific to eukaryotes, but evidence of this post-translational modification has now been discovered across all domains of life: Eucarya, Bacteria, and Archaea. In all cases, the glycans are first assembled in a step-wise manner on a polyisoprenoid carrier lipid. At some stage of lipid-linked oligosaccharide synthesis, the glycan is flipped across a membrane. Subsequently, the completed glycan is transferred to specific asparagine residues on the protein of interest. Interestingly, though the N-glycosylation pathway seems to be conserved, the biosynthetic pathways of the polyisoprenoid carriers, the specific structures of the carriers, and the glycan residues added to the carriers vary widely. In this review we will elucidate how organisms in each basic domain of life synthesize the polyisoprenoids that they utilize for N-linked glycosylation and briefly discuss the subsequent modifications of the lipid to generate a lipid-linked oligosaccharide.     

Dolichol is the major lipid component of human substantia nigra neuromelanin

Neuromelanin is a dark brown pigment present at high concentrations in dopaminergic neurones of the human substantia nigra (SN). Early electron microscopic examinations of neuromelanin fine structure revealed a significant neutral lipid component; however, the identity of this lipid has remained unknown. Here we show that the lipid component of neuromelanin pigment derived from human SN is the polyisoprenoid dolichol. Established methods were used to isolate the pigment from the SN of 32 brains and the lipid fraction was recovered in high purity and yield. Using reversed-phase HPLC, atmospheric pressure chemical ionization mass spectrometry, and 1H- and 13C-NMR techniques, we showed that the neuromelanin dolichol contained 17-23 isoprenoid units. Dolichol accounted for 14% of the mass of neuromelanin pigment; low levels of other hydrophobic compounds were detected (e.g. ubiquinone-10, alpha-tocopherol and cholesterol together accounted for < 0.5% of the neuromelanin lipid mass). This is the first time that dolichol has been identified in such a physiological setting and significantly advances our understanding of neuromelanin pigment structure and biosynthetic pathways. Furthermore, these studies identify a potential novel role for the isoprenoid pathway in the regulation of neuromelanin function and neurodegeneration within the SN.