Enzymatic features of serotonin biosynthetic enzymes and serotonin biosynthesis in plants

Serotonin, a pineal hormone in mammals, is found in a wide range of plant species at detection levels from a few nanograms to a few milligrams, and has been implicated in several physiological roles, such as flowering, morphogenesis and adaptation to environmental changes. Serotonin synthesis requires two enzymes, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H), with TDC serving as a rate-limiting step because of its high K_m relation to the substrate tryptophan (690 µM) and its undetectable expression level in control plants. However, T5H and downstream enzymes, such as serotonin N-hydroxycinnamoyl transferase (SHT), have low K_m values with corresponding substrates. This suggests that the biosynthesis of serotonin or serotonin-derived secondary metabolites is restricted to cellular stages when high tryptophan levels are present.Key words: feruloylserotonin, serotonin, tryptamine, tryptamine 5-hydroxylase, tryptophan, tryptophan biosynthesis, tryptophan decarboxylaseSerotonin is found in a broad range of plants and is abundant in reproductive organs, such as fruits and seeds.^1–3 Even though many physiological roles for serotonin in plants have been proposed,^2–7 its actual roles have yet to be examined in detail using molecular, biochemical and genetic approaches. In plants, serotonin is synthesized by two enzymes: tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H). TDC decarboxylates tryptophan into tryptamine, after which T5H hydroxylates tryptamine into serotonin.^8–10 TDC expresses at an undetectable level in rice leaves, whereas T5H expresses constitutively.^11,12     

Reconstitution of the two terminal enzymes of the heme biosynthetic pathway into phospholipid vesicles

Purified mouse protoporphyrinogen oxidase (EC 1.3.3.4) and ferrochelatase (EC 4.99.1.1), the two terminal enzymes of the heme biosynthetic pathway, have been reconstituted into phospholipid vesicles, and the kinetics of the enzymes in the reconstituted systems were compared with the values obtained with the free enzymes. The apparent Km for free protoporphyrinogen oxidase in detergent solution is 5.61 +/- 0.62 microM for free protoporphyrinogen. The Km was lower when the enzyme was inserted into phospholipid vesicles (0.78 +/- 0.28 microM) and when both enzyme and substrate were incorporated into phospholipid vesicles (0.61 +/- 0.14 microM). In the presence of cardiolipin, a phospholipid present mainly in the inner mitochondrial membrane, the value of the Km for the substrate decreased 3-fold (0.20 +/- 0.02 microM). For reconstituted ferrochelatase similar kinetic analyses were carried out and it was found that the apparent Km values were only weakly affected by the lipid environment. Studies on the orientation of ferrochelatase demonstrated that approximately 50% of the enzyme in the reconstituted system had the active site located in the inner face of the phospholipid vesicle. This is in contrast to intact mitochondria where the active site is located on the matrix side of the inner mitochondrial membrane. The activation energies for both enzymes were determined for free and reconstituted enzymes. It was found that for both enzymes the activation energies were lower for the reconstituted systems than for the free enzymes.     

Oxysterol biosynthetic enzymes

Oxysterols, herein defined as derivatives of cholesterol with a hydroxyl group on the side chain, play several roles in lipid metabolism. Members of this class regulate the expression of genes that participate in both sterol and fat metabolism, serve as substrates for the synthesis of bile acids, and are intermediates in the transfer of sterols from the periphery to the liver. Three abundant naturally occurring oxysterols are 24-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. The cholesterol hydroxylase enzymes that synthesize each of these have been isolated over the last several years and their study has produced insight into the biology of oxysterols. This article focuses on the properties of these enzymes.     

Evolution of NAD biosynthetic enzymes

Two research groups have solved crystal structures of nicotinic acid phosphoribosyltransferase (PRTase) and made the argument that PRTases in three distinct pathways of nicotinamide adenine dinucleotide (NAD) biosynthesis evolved from a common ancestor (Shin et al., 2005 and Chappie et al., 2005).     

Nuclear localization of tetrahydrobiopterin biosynthetic enzymes

Biosynthesis of the tetrahydrobiopterin (BH(4)) cofactor, essential for catecholamines and serotonin production and nitric oxide synthase (NOS) activity, requires the enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), and sepiapterin reductase (SR). Upon studying the distribution of GTPCH and PTPS with polyclonal immune sera in cross sections of rat brain, prominent nuclear staining in many neurons was observed besides strong staining in peri-ventricular structures. Furthermore, localization studies in transgenic mice expressing a Pts-LacZ gene fusion containing the N-terminal 35 amino acids of PTPS revealed beta-galactosidase in the nucleus of neurons. In contrast, PTPS-beta-galactosidase was exclusively cytoplasmic in the convoluted kidney tubules but nuclear in other parts of the nephron, indicating again that nuclear targeting may occur only in specific cell categories. Furthermore, the N terminus of PTPS acts as a domain able to target the PTPS-beta-galactosidase fusion protein to the nucleus. In transiently transfected COS-1 cells, which do not express GTPCH and PTPS endogenously, we found cytoplasmic and nuclear staining for GTPCH and PTPS. To further investigate nuclear localization of all three BH(4)-biosynthetic enzymes, we expressed Flag-fusion proteins in transiently transfected COS-1 cells and analyzed the distribution by immunolocalization and sub-cellular fractionation using anti-Flag antibodies and enzymatic assays. Whereas 5-10% of total GTPCH and PTPS and approximately 1% of total SR were present in the nucleus, only GTPCH was confirmed to be an active enzyme in nuclear fractions. The in vitro studies together with the tissue staining corroborate specific nuclear localization of BH(4)-biosynthetic proteins with yet unknown biological function.     

Fluorimetric detection of phospholipid vesicles bound to planar phospholipid membranes.

The first step in the fusion of two phospholipid membranes culminates in the aggregation of the two lipid bilayers. We have used a custom-built fluorimeter to detect multilamellar vesicles (liposomes) containing the fluorescent dye, 6-carboxyfluorescein (6-CF), bound to a planar lipid bilayer (BLM). Liposomes were added to one side of the BLM, and unbound vesicles were perfused out. This left a residual fluorescence from the BLM, but only when the membranes contained anionic lipids, and then only when millimolar levels of calcium were present. This residual fluorescence was consistently detected only when calcium was included in the buffer during the perfusion. This residual fluorescence originated from liposomes bound to the BLM. Breaking the BLM or lysing the adsorbed vesicles with distilled water abolished it. free 6-CF and/or calcium in the absence of liposomes resulted in no residual fluorescence. No residual fluorescence was detected when both the liposomes and the BLM were composed entirely of zwitterionic lipids. This was found to result from the insensitivity of the fluorimeter to a small number of liposomes adsorbed to the BLM. For this system, we conclude that calcium is necessary for both the initiation and maintenance of the state in which the vesicle membrane is bound to the planar bilayer when the membranes contain negatively charged lipids. This attachment is stronger than the interaction between zwitterionic membranes.     

Abnormalities in mitochondria-associated membranes and phospholipid biosynthetic enzymes in the mnd/mnd mouse model of neuronal ceroid lipofuscinosis

Endoplasmic reticulum-like membranes (MAM) that are associated with mitochondria have been implicated as intermediates in the import of lipids, particularly phosphatidylserine, from the endoplasmic reticulum to mitochondria (Vance, J.E. (1990) J. Biol. Chem. 265, 7248–7256; Shiao, Y.-J. et al. (1995) J. Biol. Chem. 270, 11190–11198). We have now examined further the role of MAM in lipid metabolism using the mnd/mnd mouse, a model for the human degenerative disease neuronal ceroid lipofuscinosis. The biochemical phenotype of the mnd/mnd mutant mouse (in which lipids and proteins accumulate abnormally in storage bodies in cells of affected tissues) suggested that the mutation might lead to impaired mitochondrial import of lipids and proteins as a result of a defective linkage between MAM and mitochondria. We, therefore, investigated the status of MAM and phospholipid metabolism in mnd/mnd mice livers. Separation of MAM from livers of older, but not younger, mnd/mnd mice was aberrant. In addition, the amount of the MAM-specific protein, phosphatidylethanolamine N-methyltransferase-2 (PEMT2), was greatly reduced in homogenates and MAM from livers of mnd/mnd mice of all ages, although PEMT2 mRNA abundance was normal. Moreover, PEMT activity in MAM from mnd/mnd mice was 60% less than in control mice. Activities of two additional phospholipid biosynthetic enzymes — CTP:phosphocholine cytidylyltransferase and phosphatidylserine synthase — were also reduced by >50% in mnd/mnd microsomes. Radiolabeling experiments in hepatocytes indicated that neither the mitochondrial import nor the subsequent metabolism of phosphatidylserine was grossly affected in mnd/mnd mice. However, 3 proteins (cytochrome b₅, NADH:cytochrome b₅ reductase and mitochondrial F₁F₀-ATP synthase c subunit) which are normally present in mitochondria were partially redistributed to microsomes in mnd/mnd mouse liver. These studies indicate that MAM are defective in the mnd/mnd mutant mouse in which the biochemical phenotype includes an abnormal accumulation of lipids and proteins in storage bodies.     

Enzymatic degradation and partial biosynthetic reconstitution of microsomal and mitochondrial membranes.

Guinea pig liver microsomal and mitochondrial membranes were degraded with phospholipase C and D followed by partial biosynthetic reconstitution. Activities of phosphatidylinositol synthetase in microsomal membranes and NADPH-cytochrome c reductase were almost completely lost after phospholipase C and D treatment; almost complete restoration of the original activity was achieved after biosynthesis of phosphatidylcholine in degraded microsomes, but was not reparable after biosynthesis of cytidinediphosphodiglycerides (CDP-diglycerides). The mitochondrial biosynthesis of polyglycerophosphatides was completely retained after degradation of these membranes with phospholipase C, but after similar treatment with phospholipase D, only about one-quarter of the original activity remained, the relative composition of polyglycerophosphatides being significantly different. The activity of NADPH-cytochrome c reductase of microsomes represented about 76% of the original activity after phospholipase C treatment, but only approximately 1% after treatment with phospholipase D. Although this activity could not be restored with CDP-diglyceride synthesis, it was restored to about 75% of the original activity after the biosynthesis of phosphatidylcholine in these fragments. These and additional experimental findings are discussed in terms of the relation between structural organization of lipids and proteins and enzymatic activities of membrane-bound phospholipid-synthesizing enzymes in microsomal and mitochondrial membranes isolated from guinea pig liver.     

Pyrimidine biosynthetic enzymes in Babesia hylomysci

Gero A. M. and Coombs G. H. 1982. Pyrimidine biosynthetic enzymes in Babesia hylomysci. International Journal for Parasitology12: 377–382. The enzymes that catalyse the conversion of carbamylaspartate to UMP have been demonstrated in the rodent piroplasm, Babesia hylomysci and partially characterised. They were shown to be similar to the corresponding mammalian enzymes in that dihydroorotase, orotate phosphoribosytransferase and orotidine-5'-phosphate decarboxylase were soluble, whilst dihydroorotate dehyrogenase was membrane bound and appeared to be associated with a respiratory chain. Dihydroorotate dehydrogenase was found to have a K_m (l-DHO) of 21 μm and a pH optimum of 7.8. It was inhibited by analogs of ubiquinone and several pyrimidines, dihydroazaorotate being the most effective ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 17 μ}\text{m}$). It is concluded that Babesia parasites contain a functional de novo biosynthetic pathway for pyrimidines which provides a potential target at which to direct putative chemotherapeutic agents.     

Enzymatic detection of urinary conjugated steroids after gel chromatography

An enzymatic detection method is described for urinary conjugated steroids after chromatographic fractionation with Sephadex G-25. The principle of the method is as follows. Part of a 24-h urine sample, (1–2 ml of urine) is applied directly, to a short column of Sephadex G-25 and eluted with acetate buffer solution. Steroid conjugates in each fraction are hydrolyzed with steroid sulfatase—β-glucuronidase. After enzymatic hydrolysis, an enzymatic color development reagent for steroids, either 3α-hydroxysteroid dehydrogenase or 3β-hydroxysteroid oxidase, are added and the dye formed is measured spectrophotometrically. Excretion patterns of steroid-3β-sulfates, and steroid-3α-glucoronides and steroid-3α-sulfates are shown with some patients' samples. A precision of the assay values for steroid-3α-glucuronide, steroid-3α-sulfate and steroid-3β-sulfates in urine samples and assay values for normal subjects are also studied.This simple enzymatic method for detecting the excreption patterns of urinary conjugated steroids may have a diagnostic value for clinical tests.     

Synthesis and trafficking of alkaloid biosynthetic enzymes

The biosynthesis of plant natural products involves a large number of enzymes that create and elaborate a bewildering array of chemical structures, which are generally involved in ecophysiological interactions. Alkaloids are one of the largest groups of natural products and are generally produced through an assortment of intricate pathways. The application of molecular biochemical approaches to investigate the cell biology of alkaloid pathways has revealed a paradigm for the complex, yet highly ordered, organization of biosynthetic enzymes at both the cellular and subcellular levels. Many different cell types have been implicated in alkaloid formation and storage, in one case suggesting the intercellular transport of enzymes. The localization of enzymes to numerous cellular compartments shows the importance of protein targeting in the assembly of alkaloid pathways. Recent studies have also pointed to the possible interaction of biosynthetic enzymes in multi-enzyme complexes. These processes must be considered to be integral components of the mechanisms that regulate alkaloid biosynthesis and perhaps other natural product pathways.     

Directed evolution of enzymes and biosynthetic pathways

Directed evolution is an important tool for overcoming the limitations of natural enzymes as biocatalysts. Recent advances have focused on applying directed evolution to a variety of enzymes, such as epoxide hydrolase, glyphosate N-acetyltransferase, xylanase and phosphotriesterase, in order to improve their activity, selectivity, stability and solubility. The focus has also shifted to manipulating biosynthetic pathways for the production of many naturally synthesized compounds, as well as the production of novel 'unnatural' compounds. A combined directed evolution and computational design approach is becoming increasingly important in exploring enzyme sequence-space and creating improved or novel enzymes. Fueled by recent breakthroughs in genomics and metagenomics, these developments should help expand the use of biocatalysts in industry.     

Catharanthus biosynthetic enzymes: the road ahead

Alkaloids are one of the most diverse groups of secondary metabolites found in living organisms. The most economically important alkaloids are the bisindole vinblastine, and vincristine. Unraveling the complexity of the genetic, catalytic and transport processes of monoterpene indole alkaloids biosynthesis is one of the most stimulating intellectual challenges in the plant secondary metabolism field. More than 50 metabolic steps are required to synthesize the most important alkaloids in Catharanthus roseus. Until now about only 20 of the 50 enzymes required for their biosynthesis have been determined and characterized. Hence, there are still a number of important enzymes that need to be characterized, beginning with the isolation and cloning of genes. It is also of fundamental importance to elucidate the regulatory aspects of their biosynthesis, both at the cellular and the molecular level, in order to address the question of their function in the plants that are producing them. In this review, we present an analysis of the state of the art related to the biosynthesis of the monoterpene indole alkaloids.     

Ontogeny of glycerolipid biosynthetic enzymes in swine liver and adipose tissue.

Enzymes associated with glycerolipid biosynthesis were examined in microsomal fractions of liver and adipose tissue obtained from swine of various ages. Generally, liver glycerophosphate acyltransferase, phosphatidate phosphohydrolase, diglyceride acyltransferase, and choline phosphotransferase activities were substantial at birth but increased 2- to 3-fold by day 14 postpartum, decreased at day 25, then increased at the oldest ages studied (up to 155 days postpartum). In adipose tissue, enzyme activities were low at birth and developed through day 25 in a pattern generally similar to that observed in liver. In contrast to liver, the adipose enzymes were depressed immediately postweaning (day 32) with subsequent recovery. The observed decline in adipose tissue enzyme activities expressed on a tissue basis at older ages was primarily the result of increased adipocyte size, since the activities expressed on a cell basis did not decline as rapidly. In both liver and adipose tissue, phosphatidate was the major glycerolipid synthesized by the microsomal glycerophosphate acyltransferase enzymes at all ages (generally greater than 75%). The ratio of neutral lipids to phospholipids produced by acylation of glycerophosphate was increased when a microsomal--cytosolic preparation was used as a source of enzyme in contrast to a microsomal preparation.     

Regulatory mutants and control of cysteine biosynthetic enzymes in Salmonella typhimurium.

The cysB region in Salmonella typhimurium regulates in a positive manner the noncontiguous structural genes for the enzymes responsible for sulfate reduction in cysteine biosynthesis. We treated three cysB mutants with chemical mutagens and selected 81 secondary mutants in which the inability to utilize sulfate was suppressed. Growth experiments on the suppressed mutants showed that the original loss of sulfate utilization had been corrected to varying degrees and that portions of the pathway had been established in abnormal relationship to one another. Sixty of the suppressed mutations were mapped via transductional analysis, and each was very closely linked to the original cysB mutation. We demonstrated that the cysB product functions in the regulation of the cysteine biosynthetic enzymes during both logarithmic growth and stationary phase. Mutation can alter the regulatory response of one enzyme in either an upward or downward direction while the regulation of other enzymes in the pathway remains unchanged. These data are consistent with the idea of a multivalent or multisite regulator molecule.