Sinapate esters in brassicaceous plants: biochemistry, molecular biology, evolution and metabolic engineering

Brassicaceous plants are characterized by a pronounced metabolic flux toward sinapate, produced by the shikimate/phenylpropanoid pathway, which is converted into a broad spectrum of O-ester conjugates. The abundant sinapate esters in Brassica napus and Arabidopsis thaliana reflect a well-known metabolic network, including UDP-glucose:sinapate glucosyltransferase (SGT), sinapoylglucose:choline sinapoyltransferase (SCT), sinapoylglucose:l-malate sinapoyltransferase (SMT) and sinapoylcholine (sinapine) esterase (SCE). 1-O-Sinapoylglucose, produced by SGT during seed development, is converted to sinapine by SCT and hydrolyzed by SCE in germinating seeds. The released sinapate feeds via sinapoylglucose into the biosynthesis of sinapoylmalate in the seedlings catalyzed by SMT. Sinapoylmalate is involved in protecting the leaves against the deleterious effects of UV-B radiation. Sinapine might function as storage vehicle for ready supply of choline for phosphatidylcholine biosynthesis in young seedlings. The antinutritive character of sinapine and related sinapate esters hamper the use of the valuable seed protein of the oilseed crop B. napus for animal feed and human nutrition. Due to limited variation in seed sinapine content within the assortment of B. napus cultivars, low sinapine lines cannot be generated by conventional breeding giving rise to genetic engineering of sinapate ester metabolism as a promising means. In this article we review the progress made throughout the last decade in identification of genes involved in sinapate ester metabolism and characterization of the encoded enzymes. Based on gene structures and enzyme recruitment, evolution of sinapate ester metabolism is discussed. Strategies of targeted metabolic engineering, designed to generate low-sinapate ester lines of B. napus, are evaluated.     

Plant lectins: occurrence,biochemistry, functions and applications

Growing insights into the many roles of glycoconjugates in biorecognition as ligands for lectins indicates a need to compare plant and animal lectins. Furthermore, the popularity of plant lectins as laboratory tools for glycan detection and characterization is an incentive to start this review with a brief introduction to landmarks in the history of lectinology. Based on carbohydrate recognition by lectins, initially described for concanavalin A in 1936, the chemical nature of the ABH-blood group system was unraveled, which was a key factor in introducing the term lectin in 1954. How these versatile probes are produced in plants and how they are swiftly and efficiently purified are outlined, and insights into the diversity of plant lectin structures are also given. The current status of understanding their functions calls for dividing them into external activities, such as harmful effects on aggressors, and internal roles, for example in the transport and assembly of appropriate ligands, or in the targeting of enzymatic activities. As stated above, attention is given to intriguing parallels in structural/functional aspects of plant and animal lectins as well as to explaining caveats and concerns regarding their application in crop protection or in tumor therapy by immunomodulation. Integrating the research from these two lectin superfamilies, the concepts are discussed on the role of information-bearing glycan epitopes and functional consequences of lectin binding as translation of the sugar code (functional glycomics).     

Sulfur-driven autotrophic denitrification: diversity, biochemistry, and engineering applications

Sulfur-driven autotrophic denitrification refers to the chemolithotrophic process coupling denitrification with the oxidation of reduced inorganic sulfur compounds. Ever since 1904, when Thiobacillus denitrificans was isolated, autotrophic denitrifiers and their uncultured close relatives have been continuously identified from highly diverse ecosystems including hydrothermal vents, deep sea redox transition zones, sediments, soils, inland soda lakes, etc. Currently, 14 valid described species within α-, β-, γ-, and ε-Proteobacteria have been identified as capable of autotrophic denitrification. Autotrophic denitrification is also widely applied in environmental engineering for the removal of sulfide and nitrate from different water environments. This review summarizes recent researches on autotrophic denitrification, highlighting its diversity, metabolic traits, and engineering applications.     

Lack of regulation of aromatic L-amino acid decarboxylase in intact bovine chromaffin cells

Aromatic l-amino acid decarboxylase (AADC) is the second enzyme in the catecholamine biosynthetic pathway, and its activity is generally considered not to be limiting, and therefore not involved, in regulating flux through this pathway. Recent studies showing that its activity can be regulated in vivo and that the enzyme can be phosphorylated and activated in vitro have raised the possibility that AADC may play more than an obligatory role in catecholamine biosynthesis. In the present study, the phosphorylation and activity of AADC was evaluated relative to that of tyrosine hydroxylase (TH; the first and rate-limiting enzyme in the pathway) in intact bovine chromaffin cells. Treatment of chromaffin cells with elevated potassium, acetylcholine, phorbol dibutyrate, forskolin, or okadaic acid each increased 32P incorporation into TH (after metabolic labeling of ATP pools with 32P(i)) and TH activity. In contrast, as measured in matched samples, 32P incorporation into AADC was not detected and none of the treatments altered AADC activity. Thus, that AADC can be phosphorylated and activated in vitro has questionable physiological significance.     

Catechol-O-methyltransferase and aromatic L-amino acid decarboxylase activities in human gastrointestinal tissues

Human gastrointestinal samples from the corpus, antrum, bulbus, jejunum and ileum were assayed for soluble and membrane-bound catechol-O-methyltransferase (COMT) and aromatic L-amino acid decarboxylase (AADC) activity in vitro. The mean soluble COMT activities with 3,4-dihydroxybenzoic acid (DBA) and 3,4-dihydroxyphenylalanine (L-DOPA) as substrate were 70-242 and 70-174 pmol/min mg, respectively. The membrane-bound COMT activities ranged from 33 to 60 pmol/min mg in the different parts of the intestine. The AADC activities, measured with L-DOPA as the substrate, increased from 114 pmol/min mg in the corpus to 3488 pmol/min mg in the jejunum. The affinity of the soluble COMT was approximately 20 times higher for DBA (Km 15-19 microM) than for L-DOPA (Km 300-600 microM). The Km-values for L-DOPA of AADC and COMT were of the same order of magnitude. The specific COMT inhibitors, nitecapone and OR-611, effectively inhibited in vitro the human intestinal COMT activity. Nanomolar concentrations caused 50% inhibition with both DBA and L-DOPA as substrate.     

D-amino acid oxidase,aromatic L-amino aminotransferase,and aromatic lactate dehydrogenase from several yeast species: Comparison of enzyme activities and enzyme specificities

Production of D-amino acid oxidase, L-aromatic aminotransferase and aromatic lactate dehydrogenase by several yeast species was examined. Of 16 strains tested, Trigonopsis variabilis and Rhodosporidium toruloides were found to be most suitable for D-amino acid oxidase production, T. variabilis and Brettanomyces anomalus for L-aromatic aminotransferase production, and Hansenula polymorpha, Cryptococcus terreus, and Candida maltosa for aromatic lactate dehydrogenase production. This selection is based on a high amount of enzyme activity as well as a broad enzyme specificity. The data will be reported here.     

The Sleeping Beauty transposable element: evolution, regulation and genetic applications

Members of the Tc1/mariner superfamily of transposable elements isolated from vertebrate species are inactive due to the accumulation of mutations. A representative of a subfamily of fish elements estimated to be last active > 10 million years ago has been reconstructed, and named Sleeping Beauty(SB). This element opened up new avenues for studies on DNA transposition in vertebrates, and for the development of transposon tools for genetic manipulation in important model species and in humans. Multiple transposase binding sites within the terminal inverted repeats, a transpositional enhancer sequence, unequal affinity of the transposase to the binding sites and the activity of the cellular HMGB1 protein all contribute to a highly regulated assembly of SB synaptic complexes, which is likely a requirement for the subsequent catalytic steps. Host proteins involved in double-strand DNA break repair are limiting factors of SB transposition in mammalian cells, underscoring evolutionary, structural and functional links between DNA transposition, retroviral integration and V(D)J recombination. SB catalyzes efficient cut-and-paste transposition in a wide range of vertebrate cells in tissue culture, and in somatic tissues as well as the germline of the mouse and zebrafish in vivo, indicating its usefulness as a vector for transgenesis and insertional mutagenesis.     

Biosensors and their applications in microbial metabolic engineering

Many metabolic pathways in microbial hosts have been created, modified and engineered to produce useful molecules. The titer and yield of a final compound is often limited by the inefficient use of cellular resources and imbalanced metabolism. Engineering sensory-regulation devices that regulate pathway gene expression in response to the environment and metabolic status of the cell have great potential to solve these problems, and enhance product titers and yields. This review will focus on recent developments in biosensor design, and their applications for controlling microbial behavior.     

Isolation and characterization of a cDNA clone encoding human aromatic L-amino acid decarboxylase

The nucleotide sequence of a cDNA clone that includes the entire coding region of human aromatic L-amino acid decarboxylase gene is presented. A human pheochromocytoma cDNA library was screened using an oligonucleotide probe which corresponded to a partial amino acid sequence of the enzyme purified from the human pheochromocytoma. The isolated cDNA clone encoded a protein of 480 amino acids with a calculated molecular mass of 53.9 kDa. The amino acid sequence Asn-Phe-Asn-Pro-His-Lys-Trp around a possible cofactor (pyridoxal phosphate) binding site is identical in human, Drosophila, and pig enzymes.     

基因敲除技术及其在微生物代谢工程方面的应用

应用基因工程技术对微生物细胞内的代谢通量进行重新设计,是获得高产高效的工业菌株的重要手段之一。基因敲除是20世纪80年代发展起来的一项重要的分子生物学技术,利用基因敲除技术可阻断细胞的代谢旁路。或通过引入突变位点改变目的产物的产量或质量而达到调节代谢流,优化代谢途径的目的。简单介绍了基因敲除技术的操作过程,重点讨论基因敲除技术的敲除策略及在微生物代谢工程方面的应用,并展望了相关技术在诸多领域的发展趋势。     

Phosphorylation and activation of brain aromatic L-amino acid decarboxylase by cyclic AMP-dependent protein kinase

Aromatic L-amino acid decarboxylase (AAAD), an enzyme required for the synthesis of catecholamines, indoleamines, and trace amines, is rapidly activated by cyclic AMP-dependent pathways in striatum and midbrain in vivo, suggesting enzyme phosphorylation. We now report that the catalytic subunit of cyclic AMP-dependent protein kinase (PKA) directly phosphorylated AAAD immunoprecipitated from homogenates prepared from the mouse striatum and midbrain in vitro. Under the same phosphorylation conditions, the catalytic subunit of PKA also phosphorylated a recombinant AAAD protein expressed in Escherichia coli transfected with an AAAD cDNA isolated from the bovine adrenal gland. The PKA-induced AAAD phosphorylation of immunoprecipitates from striatum and midbrain was time and concentration dependent and blocked by a specific PKA peptide inhibitor. Incubation of the catalytic subunit of PKA with striatal homogenates increased enzyme activity by approximately 20% in a time- and concentration-dependent manner. Moreover, incubation of the catalytic subunit of PKA with recombinant AAAD increased activity by approximately 70%. A direct phosphorylation of AAAD protein by PKA might underlie the cyclic AMP-induced rapid and transient activation of AAAD in vivo.     

Phosphatidylinositol biosynthesis: biochemistry and regulation

Phosphatidylinositol (PI) is a ubiquitous membrane lipid in eukaryotes. It is becoming increasingly obvious that PI and its metabolites play a myriad of very diverse roles in eukaryotic cells. The Saccharomyces cerevisiae PIS1 gene is essential and encodes PI synthase, which is required for the synthesis of PI. Recently, PIS1 expression was found to be regulated in response to carbon source and oxygen availability. It is particularly significant that the promoter elements required for these responses are conserved evolutionarily throughout the Saccharomyces genus. In addition, several genome-wide strategies coupled with more traditional screens suggest that several other factors regulate PIS1 expression. The impact of regulating PIS1 expression on PI synthesis will be discussed along with the possible role(s) that this may have on diseases such as cancer.     

Bacterial dehalogenases: biochemistry, genetics, and biotechnological applications.

This review is a survey of bacterial dehalogenases that catalyze the cleavage of halogen substituents from haloaromatics, haloalkanes, haloalcohols, and haloalkanoic acids. Concerning the enzymatic cleavage of the carbon-halogen bond, seven mechanisms of dehalogenation are known, namely, reductive, oxygenolytic, hydrolytic, and thiolytic dehalogenation; intramolecular nucleophilic displacement; dehydrohalogenation; and hydration. Spontaneous dehalogenation reactions may occur as a result of chemical decomposition of unstable primary products of an unassociated enzyme reaction, and fortuitous dehalogenation can result from the action of broad-specificity enzymes converting halogenated analogs of their natural substrate. Reductive dehalogenation either is catalyzed by a specific dehalogenase or may be mediated by free or enzyme-bound transition metal cofactors (porphyrins, corrins). Desulfomonile tiedjei DCB-1 couples energy conservation to a reductive dechlorination reaction. The biochemistry and genetics of oxygenolytic and hydrolytic haloaromatic dehalogenases are discussed. Concerning the haloalkanes, oxygenases, glutathione S-transferases, halidohydrolases, and dehydrohalogenases are involved in the dehalogenation of different haloalkane compounds. The epoxide-forming halohydrin hydrogen halide lyases form a distinct class of dehalogenases. The dehalogenation of alpha-halosubstituted alkanoic acids is catalyzed by halidohydrolases, which, according to their substrate and inhibitor specificity and mode of product formation, are placed into distinct mechanistic groups. beta-Halosubstituted alkanoic acids are dehalogenated by halidohydrolases acting on the coenzyme A ester of the beta-haloalkanoic acid. Microbial systems offer a versatile potential for biotechnological applications. Because of their enantiomer selectivity, some dehalogenases are used as industrial biocatalysts for the synthesis of chiral compounds. The application of dehalogenases or bacterial strains in environmental protection technologies is discussed in detail.     

MicroRNA-mediated gene regulation: potential applications for plant genetic engineering

Food security is one of the most important issues challenging the world today. Any strategies to solve this problem must include increasing crop yields and quality. MicroRNA-based genetic modification technology (miRNA-based GM tech) can be one of the most promising solutions that contribute to agricultural productivity directly by developing superior crop cultivars with enhanced biotic and abiotic stress tolerance and increased biomass yields. Indirectly, the technology may increase usage of marginal soils and decrease pesticide use, among other benefits. This review highlights the most recent progress of transgenic studies utilizing various miRNAs and their targets for plant trait modifications, and analyzes the potential of miRNA-mediated gene regulation for use in crop improvement. Strategies for manipulating miRNAs and their targets in transgenic plants including constitutive, stress-induced, or tissue-specific expression of miRNAs or their targets, RNA interference, expressing miRNA-resistant target genes, artificial target mimic and artificial miRNAs were discussed. We also discussed potential risks of utilizing miRNA-based GM tech. In general, miRNAs and their targets not only provide an invaluable source of novel transgenes, but also inspire the development of several new GM strategies, allowing advances in breeding novel crop cultivars with agronomically useful characteristics.     

Cholesterol-5,6-epoxides: Chemistry,biochemistry, metabolic fate and cancer

In the nineteen sixties it was proposed that cholesterol might be involved in the etiology of cancers and cholesterol oxidation products were suspected of being causative agents. Researchers had focused their attention on cholesterol-5,6-epoxides (5,6-ECs) based on several lines of evidence: 1) 5,6-ECs contained an oxirane group that was supposed to confer alkylating properties such as those observed for aliphatic and aromatic epoxides. 2) cholesterol-5,6-epoxide hydrolase (ChEH) was induced in pre-neoplastic lesions of skin from rats exposed to ultraviolet irradiations and ChEH was proposed to be involved in detoxification processes like other epoxide hydrolases. However, 5,6-ECs failed to induce carcinogenicity in rodents which ruled out a potent carcinogenic potential for 5,6-ECs. Meanwhile, clinical studies revealed an anomalous increase in the concentrations of 5,6β-EC in the nipple fluids of patients with pre-neoplastic breast lesions and in the blood of patients with endometrious cancers, suggesting that 5,6-ECs metabolism could be linked with cancer. Paradoxically, ChEH has been recently shown to be totally inhibited by therapeutic concentrations of tamoxifen (Tam), which is one of the main drugs used in the hormonotherapy and the chemoprevention of breast cancers. These data would suggest that the accumulation of 5,6-ECs could represent a risk factor, but we found that 5,6-ECs were involved in the induction of breast cancer cell differentiation and death induced by Tam suggesting a positive role of 5,6-ECs. These observations meant that the biochemistry and the metabolism of 5,6-ECs needed to be extensively studied. We will review the current knowledge and the future direction of 5,6-ECs chemistry, biochemistry, metabolism, and relationship with cancer.     

A single gene codes for aromatic L-amino acid decarboxylase in both neuronal and non-neuronal tissues

We have sought to determine whether aromatic L-amino acid decarboxylase which functions as a neurotransmitter biosynthetic enzyme in neuronal cells can be distinguished from an enzyme with similar activity found in peripheral tissues where no neurotransmitters are synthesized. Aromatic L-amino acid decarboxylase was purified to electrophoretic homogeneity from bovine adrenal medulla, and highly specific antibodies were produced. In addition, a DNA clone complementary to aromatic L-amino acid decarboxylase mRNA was isolated by immunological screening of a lambda gt11 cDNA expression library. We have used these antibodies and cDNA probes for biochemical, immunochemical, and molecular analyses. A single form of aromatic L-amino acid decarboxylase is detected in rat and bovine tissue. Specifically, aromatic L-amino acid decarboxylase protein is biochemically and immunochemically indistinguishable in brain, liver, kidney, and adrenal medulla. Hybridization to aromatic L-amino acid decarboxylase cDNA identifies a single mRNA species of 2.3 kilobase pairs in rat tissue. Furthermore, Southern blot analysis reveals that a single gene codes for aromatic L-amino acid decarboxylase.     

Purification and properties of aromatic L-amino acid decarboxylase (4.1.1.28) of rat brain]

L-aromatic aminoacid decarboxylase has been purified more than thousand times from homogenates of rat brain, in several steps : centrifugation, DEAE-cellulose, CM cellulose, hydroxylapatite, DEAE sephadex. Its properties have been studied, most of them on an intermediate fraction of the purification, because of the instability of the purified enzyme in spite of the addition of different stabilizing agents : the enzyme decarboxylates 5-hydroxytryptophan (5 HTP) and DOPA in a ratio constant throughout the purification but does not decarboxylate tryptophan, tyrosine, histidine at a measurable rate. Optimum pH, Km, Vm, have been measured with 5 HTP and DOPA as substrates. The enzyme has a molecular weight of 115.000, an apparent isoelectric point of 6,4-6,5. It is inhibited by serotonin, dopamine, some cations : Cu++, Fe++, Ni++ by N-ethylmaleimide, sodium dodecylsulfate. Some pyridoxal-5 phosphate (PLP) remains strongly bound to the enzyme. For relatively weak concentrations of substrate, the enzyme is inhibited by an excess of PLP ; for weak concentrations of PLP, the enzyme in inhibited by an excess of substrate, particularly of DOPA. We also observe a spontaneous decarboxylation of the substrates that reaches a plateau and is enhanced by high concentrations of PLP, by serotonin, dopamine, Cu++ and reduced by mercaptoethanol and the presence of crude or boiled homogenates. Several possible explanations of the spontaneous decarboxylation and of the enzymic inhibitions by an excess of PLP and by the substrates are given.