Cloning and characterization of DOPA decarboxylase in Litopenaeus vannamei and its roles in catecholamine biosynthesis, immunocompetence, and antibacterial defense by dsRNA-mediated gene silencing.

Catecholamines (CAs) play critical roles in regulating physiological and immunological homeostasis in invertebrates and vertebrates under stressful environments. DOPA decarboxylase (DDC), an enzyme responsible for the decarboxylation step of dopamine synthesis, participates in neurotransmitter metabolism and innate immunity. In shrimp, two genes encoding CA-related enzymes, tyrosine hydroxylase and dopamine beta-hydroxylase, were further identified and characterized as neuroendocrine-immune regulators. In this study, full-length complementary DNA of DDC cloned from the thoracic ganglia of shrimp, Litopenaeus vannamei, (LvDDC) was predicted to encode a 452-amino acid protein with a pyridoxal-dependent decarboxylase-conserved domain, and this deduced protein of LvDDC was phylogenetically closely related to insect DDC. LvDDC messenger RNA expression was analyzed by a semiquantitative RT-PCR and a real-time quantitative RT-PCR and found to be abundant in the hepatopancreas and nervous system but at low levels in haemocytes, heart, stomach, and gills. To determine the role of LvDDC, double-stranded (ds)RNA was used for in vivo assessments. LvDDC-depleted shrimp revealed significant increases in the total haemocyte count, hyaline cells, granular cells, phenoloxidase activity, and respiratory bursts of haemocytes per unit of haemolymph, and phagocytic activity and clearance efficiency toward Vibrio alginolyticus. Further, decreased LvDDC mRNA expression was accompanied by decreases in dopamine, glucose, and lactate levels in haemolymph. In shrimp that received LvDDC-dsRNA for 3 days and were then challenged with V. alginolyticus, the survival rate of LvDDC-depleted shrimp was significantly higher than that of shrimp that received diethyl pyrocarbonate-water or non-targeted dsRNA. In conclusion, the cloned LvDDC was responsible for controlling dopamine synthesis, which then regulated physiological and immune responses in L. vannamei.

Molecular insights into the pathogenicity of variants associated with the aromatic amino acid decarboxylase deficiency.

Dopa decarboxylase (DDC or AADC) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the decarboxylation of L-aromatic amino acids into the corresponding aromatic amines. AADC deficiency is an inborn error of neurotransmitters biosynthesis with an autosomal recessive inheritance. About 30 pathogenic mutations have been identified, but the enzymatic phenotypes causing AADC deficiency are unknown, and the therapeutic management is challenging. Here, we report biochemical and bioinformatic analyses of the human wild-type DDC and the pathogenic variants G102S, F309L, S147R and A275T whose mutations concern amino acid residues at or near the active site. We found that the mutations cause, even if to different extents, a decreased PLP binding affinity (in the range 1.4-170-fold), an altered state of the bound coenzyme and of its microenvironment, and a reduced catalytic efficiency (in the range 17-930-fold). Moreover, as compared to wild-type, the external aldimines formed by the variants with L-aromatic amino acids exhibit different spectroscopic features, do not protect against limited proteolysis, and lead to the formation, in addition to aromatic amines, of cyclic-substrate adducts. This suggests that these external Schiff bases are not properly oriented and anchored, i.e., in a conformation not completely productive for decarboxylation. The external aldimines that the variants form with D-Dopa also appear not to be correctly located at their active site, as suggested by the rate constants of PLP-L-Dopa adduct production higher than that of the wild-type. The possible therapeutic implications of the data are discussed in the light of the molecular defects of the pathogenic variants.

Identification and characterization of a novel form of the human L-dopa decarboxylase mRNA.

L-Dopa decarboxylase (DDC) has been cloned from several species and was shown to undergo alternative splicing within its 5'-untranslated and coding regions. In this report, we describe a novel splice variant of DDC mRNA in human tissue, lacking exons 10-15 of the full-length transcript but including an alternative exon 10. The isolated alternative human DDC cDNA (alt-DDC) was cloned from human placenta, and was found to be of the neuronal type. Northern blot analysis indicated that the alt-DDC transcript is expressed in high levels in human kidney. Our results demonstrate the detection of a new alternative splicing event within the coding region of the human DDC mRNA, further suggesting that the single copy human DDC gene undergoes complex processing leading to the formation of multiple mRNA isoforms.

L-DOPA decarboxylase association with membranes in mouse brain.

This work presents evidence on the association of active DDC molecules with membranes in mammalian brain. L-DOPA decarboxylase (DDC) is generally considered to be a cytosolic enzyme. Membrane-associated DDC was detected by immunoblotting and enzymatic assay experiments. DDC activity and immunoreactivity could be partially extracted from mammalian brain membranes by detergent. Fractionation of membranes by temperature-induced phase separation in Triton X-114, resulted in the recovery of membrane-associated DDC in separation phases where integral and hydrophobic membrane proteins separate. Treatment of membranes with phosphatidylinositol-specific phospholipase C or proteinase K, did not elute membrane-associated DDC activity, suggesting that a population of DDC molecules exist embedded within membranes. The elucidation of the functional significance of the enzyme's association with membranes could provide us with new information leading to the better understanding of the biological pathways that DDC is involved in.

Bacterial-injection-induced syntheses of N-beta-alanyldopamine and Dopa decarboxylase in the hemolymph of coleopteran insect, Tenebrio molitor larvae.

Injection of Escherichia coli into larvae of the coleopteran Tenebrio molitor resulted in the appearance of a dopamine-like substance on the electrochemical detector. To characterize this dopamine-like substance, we purified it to homogeneity from the immunized hemolymph and determined its molecular structure to be N-beta-alanyldopamine using the liquid chromatographic/tandem mass spectrometric method. Chemically synthesized N-beta-alanyldopamine showed the same retention time on HPLC as the purified N-beta-alanyldopamine from immunized larvae. To elucidate the molecular mechanism of N-beta-alanyldopamine synthesis in vivo, we examined the enzyme activity of Dopa decarboxylase against E. coli-injected hemolymph of T. molitor larvae. The enzyme activity of Dopa decarboxylase increased dramatically approximately 8 h after injection; Dopa decarboxylase activity of injected larvae being 10-times higher than naive larvae after 24 h. To evaluate the extent of quantitative changes of Dopa decarboxylase in response to bacterial challenge, Tenebrio Dopa decarboxylase was purified to homogeneity from the whole larvae and a cDNA clone for Tenebrio Dopa decarboxylase was isolated. RNA blot hybridization revealed that expression of the Dopa decarboxylase gene was activated transiently 3-8 h after E. coli challenge. Immunoprecipitation experiments showed that Tenebrio Dopa decarboxylase was detected from 8 to 24 h in E. coli-injected larval extract. Thus, bacterial injection into T. molitor larvae might induce transcriptional activation of a Dopa decarboxylase gene, and then synthesis of N-beta-alanyldopamine. The synthesized N-beta-alanyldopamine might be used as a substrate by phenoloxidase during melanin synthesis in the humoral defense response or the melanotic encapsulation reaction of the cellular defense response.

Reaction specificity of native and nicked 3,4-dihydroxyphenylalanine decarboxylase.

3,4-Dihydroxyphenylalanine (Dopa) decarboxylase is a stereospecific pyridoxal 5'-phosphate (PLP)-dependent alpha-decarboxylase that converts L-aromatic amino acids into their corresponding amines. We now report that reaction of the enzyme with D-5-hydroxytryptophan or D-Dopa results in a time-dependent inactivation and conversion of the PLP coenzyme to pyridoxamine 5'-phosphate and PLP-D-amino acid Pictet-Spengler adducts, which have been identified by high performance liquid chromatography. We also show that the reaction specificity of Dopa decarboxylase toward aromatic amines depends on the experimental conditions. Whereas oxidative deamination occurs under aerobic conditions (Bertoldi, M., Moore, P. S., Maras, B., Dominici, P., and Borri Voltattorni, C. (1996) J. Biol. Chem. 271, 23954-23959; Bertoldi, M., Dominici, P., Moore, P. S., Maras, B., and Borri Voltattorni, C. (1998) Biochemistry 37, 6552-6561), half-transamination and Pictet-Spengler reactions take place under anaerobic conditions. Moreover, we examined the reaction specificity of nicked Dopa decarboxylase, obtained by selective tryptic cleavage of the native enzyme between Lys334 and His335. Although this enzymatic species does not exhibit either decarboxylase or oxidative deamination activities, it retains a large percentage of the native transaminase activity toward D-aromatic amino acids and displays a slow transaminase activity toward aromatic amines. These transamination reactions occur concomitantly with the formation of cyclic coenzyme-substrate adducts. Together with additional data, we thus suggest that native Dopa decarboxylase can exist as an equilibrium among "open," "half-open," and "closed" forms.

Cloning and expression of pig kidney dopa decarboxylase: comparison of the naturally occurring and recombinant enzymes.

L-Aromatic amino acid decarboxylase (dopa decarboxylase; DDC) is a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme that catalyses the decarboxylation of L-dopa and other L-aromatic amino acids. To advance structure-function studies with the enzyme, a cDNA that codes for the protein from pig kidney has been cloned by joining a partial cDNA obtained by library screening with a synthetic portion constructed by the annealing and extension of long oligonucleotides. The hybrid cDNA was then expressed in Escherichia coli to produce recombinant protein. During characterization of the recombinant enzyme it was unexpectedly observed that it possesses certain differences from the enzyme purified from pig kidney. Whereas the later protein binds 1 molecule of PLP per dimer, the recombinant enzyme was found to bind two molecules of coenzyme per dimer. Moreover, the Vmax was twice that of the protein purified from tissue. On addition of substrate, the absorbance changes accompanying transaldimination were likewise 2-fold greater in the recombinant enzyme. Examination of the respective apoenzymes by absorbance, CD and fluorescence spectroscopy revealed distinct differences. The recombinant apoprotein has no significant absorbance at 335 nm, unlike the pig kidney apoenzyme; in the latter case this residual absorbance is associated with a positive dichroic signal. When excited at 335 nm the pig kidney apoenzyme has a pronounced emission maximum at 385 nm, in contrast with its recombinant counterpart, which shows a weak broad emission at about 400 nm. However, the holoenzyme-apoenzyme transition did not markedly alter the respective fluorescence properties of either recombinant or pig kidney DDC when excited at 335 nm. Taken together, these findings indicate that recombinant pig kidney DDC has two active-site PLP molecules and therefore displays structural characteristics typical of PLP-dependent homodimeric enzymes. The natural enzyme contains one active-site PLP molecule whereas the remaining PLP binding site is most probably occupied by an inactive covalently bound coenzyme derivative; some speculations are made about its origin. The coenzyme absorbing bands of recombinant DDC show a modest pH dependence at 335 and 425 nm. A putative working model is presented to explain this behaviour.

Purification and characterisation of L-DOPA decarboxylase from pharate pupae of Ceratitis capitata. A comparison with the enzyme purified from the white prepupae.

In this paper we describe the purification of L-DOPA decarboxylase (DDC) to homogeneity from the developmental stage just before the eclosion (pharate pupae) of Ceratitis capitata. The enzyme was found to have a mol wt of approximately 100,000 and to be composed of two identical subunits (50,000 mol wt each). Polyclonal antibodies raised against the isolated enzyme reacted with the 50,000 dalton subunit and precipitated enzyme activity. Furthermore, properties of the enzyme isolated from the pharate pupa stage, were compared with those of DDC purified from the white prepupa stage with respect to substrate specificity, response to polyclonal antibodies, behaviour towards different cations and dependence of enzyme activity on the concentration of pyridoxal phosphate.

Inhibition of pig kidney L-aromatic amino acid decarboxylase by 2,3-methano-m-tyrosines.

Both racemic (E)- and (Z)-2,3-methano-m-tyrosines (9E and 9Z) have been synthesized from a common intermediate, monoester (Z)-1-(ethoxycarbonyl)-2-[3-[(2-methoxyethoxy)methoxy]phenyl] cyclopropanecarboxylic acid (5). Quinine and ephedrine, respectively, were used to resolve their N-tert-butoxycarbonyl (Boc) derivatives. Among the compounds prepared, the (+)-(E)-diastereomer of 9 is the most potent inhibitor of L-aromatic amino acid decarboxylase (Dopa decarboxylase), having a Ki of 22 microM, with the (-)-Z-diastereomer (9Z) second at Ki = 49 microM. (+)-9E is a 45-fold more potent inhibitor of DDC than its acyclic analogue, D-m-tyrosine.

Purification of dopa decarboxylase from bovine striatum.

Pyridoxal phosphate-dependent DOPA decarboxylase has been purified from bovine striatum to a specific activity of 1.6 U/mg protein. After ammonium sulfate precipitation (30-60%) it was purified by DEAE-Sephacel, Sephacryl S-200, and TSK Phenyl 5 PW chromatography. The purified enzyme showed a single silver straining band with polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions. The bovine striatal DOPA decarboxylase is a dimer (subunit Mr = 56,000 by SDS-PAGE) with a native Mr of 106,000 as judged by chromatography on Sephacryl S-200 and by sedimentation analysis. Similar to the DOPA decarboxylase purified from non-CNS tissues, the bovine striatal enzyme requires free sulfhydryl groups for activity, is strongly inhibited by heavy metal ions, and can decarboxylate 5-hydroxytryptophan as well. It should be noted, however, that the final enzyme preparation is enriched in DOPA decarboxylase activity. The distribution of the DOPA decarboxylase and 5-HTP decarboxylase activities also varies among several bovine brain regions. In addition, heat treatment of the enzyme preparation inactivated the two decarboxylation activities at different rates.

L-dopa decarboxylase in Ceratitis capitata white puparia and human: a comparative study.

1. L-DOPA decarboxylase (DDC) from Ceratitis capitata and from human kidney have been purified by the same methodology. 2. Both enzymes show mol. wts of 100,000, consisting of two identical mol. wt subunits and solely decarboxylate L-DOPA. 3. In the presence of 5-hydroxytryptophan (5-HTP) only the DDC activity from human kidney is remarkably reduced. 4. Addition of exogenous coenzyme is essential only for human DDC activity. 5. Polyclonal antibodies, raised against DDC purified from insects or humans, cross-react with both antigens.

[Purification and partial sequencing of L-dopa decarboxylase from pheochromocytoma in rats]. / Purification et séquençage partiel de la L-dopa décarboxylase de phéochromocytome de rat.

L-DOPA decarboxylase was purified from rat pheochromocytoma. Tryptic digestion of this enzyme permitted obtaining fourteen peptides. The comparison of the sequence of L-DOPA decarboxylase from other species with one of these peptides demonstrates a great preservation of this protein.

Evidence for PQQ as cofactor in 3,4-dihydroxyphenylalanine (dopa) decarboxylase of pig kidney.

Pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase (EC 4.1.1.28) was purified to homogeneity. Treatment of the enzyme with phenylhydrazine (PH) according to a procedure developed for analysis of quinoproteins gave products which were identified as the hydrazone of pyridoxal phosphate (PLP) and the C(5)-hydrazone of pyrroloquinoline quinone (PQQ). This method failed, however, in quantifying the amounts of cofactor. Direct hydrolysis of the enzyme by refluxing with hexanol and concentrated HCl led to detachment of PQQ from the protein in a quantity of 1 PQQ per enzyme molecule. In view of the reactivity of PQQ towards amines and amino acids, we postulate that it participates as a covalently bound cofactor in the catalytic cycle of the enzyme, in interplay with PLP. Since several other enzymes have been reported to show the atypical behaviour of dopa decarboxylase, it seems that the PLP-containing group of enzymes can be subdivided into pyridoxoproteins and pyridoxo-quinoproteins.

Histidine decarboxylase from rat gastric mucosa: heterogeneity and enzyme forms modification.

Rat gastric mucosal histidine decarboxylase is shown to exist in the crude extract as three active charged forms which are separable by isoelectric focusing. The distribution of enzyme activity in the three forms is independent of the homogenizing medium and of the isoelectric focusing procedure indicating that the heterogeneity does not arise during isolation. Multiple forms correspond to histidine decarboxylase and are related neither to 3,4-dihydroxyphenylalanine decarboxylase nor to the result of aggregation. This charge difference between the enzyme forms changes according to the time of storage and to the temperature, leading to the generation of less negatively charged species. The conversion cannot be attributed to proteolytic degradation nor to differences in stability between forms. The data indicate that these alternative charged states may really result from an in vivo post-translational modification of the enzyme.

Purification and characterization of rat-liver 3,4-dihydroxyphenylalanine decarboxylase.

A simple and rapid procedure, which takes advantage of the effectiveness of conventional and HPLC hydrophobic interaction, for the isolation of highly purified rat liver 3,4-dihydroxyphenylalanine decarboxylase is described in detail. Some of its structural and functional properties are reported and discussed in comparison with those of pig kidney 3,4-dihydroxyphenylalanine decarboxylase.

Purification of L-dopa decarboxylase from rat liver and production of polyclonal and monoclonal antibodies against it.

L-DOPA decarboxylase [DDC, aromatic-L-amino acid carboxyl-lyase, EC 4.1.1.28] was purified 800-fold from rat liver by several column chromatographic steps. The enzyme (specific activity, about 6 mumol/min X mg protein) had a molecular weight of 100,000 and gave a single band with a molecular weight of 50,000 on SDS-polyacrylamide gel electrophoresis. Its isoelectric point was pH 5.7. The absorption spectrum in the visible region of the purified DDC showed maxima at 330 and 420 nm. Polyclonal and monoclonal antibodies against DDC were produced by using this purified protein as an antigen. Polyclonal anti-DDC serum immunoprecipitated the DDC activities of rat, guinea-pig and rabbit livers (about 1, 10, and more than 100 microliter of antiserum, respectively, were required for 50% precipitation of 2 nmol/min of activity of these enzymes). The monoclonal antibody, named MA-1, belonged to the IgG1 subclass and immunoprecipitated the DDC activities of rat and guinea-pig livers to the same extent (about 0.5 micrograms of IgG was required to immunoprecipitate 2 nmol/min activity of each enzyme), but it did not affect the rabbit enzyme. The antibody MA-1 detected DDC molecules of both the purified enzyme and crude homogenate of rat liver blotted onto a nitrocellulose sheet. Immunohistochemically this antibody also stained specific neurons in the substantia nigra, raphe nucleus and locus coeruleus of rat brain.

Immunohistochemical analysis of the cross-reaction of anti-rat histidine decarboxylase antibody with guinea-pig DOPA decarboxylase.

L-Histidine decarboxylase [L-histidine carboxylyase, HDC, EC 4.1.1.22] is an enzyme distinct from L-DOPA decarboxylase [L-aromatic amino acid carboxylyase, DDC, EC 4.1.1.28]: the two decarboxylases from fetal rat liver were completely separated from each other by DEAE-cellulose column chromatography and by affinity chromatography with L-carnosine as a ligand. The antibody raised against this HDC inhibited the HDC's from rat and guinea-pig brains very strongly, but their DDCs very weakly. However, in immunofluorescent histochemical studies, the antibody cross-reacted with DDC-like immunoreactive structures, such as chromaffin cells of the adrenal medulla, the raphe nucleus, the substantia nigra, and the locus coeruleus of the brain of guinea-pigs, but not of rats, suggesting that these two decarboxylases share some antigenic structures.