Induction of dopa (3,4-dihydroxyphenylalanine) decarboxylase in blowfly integument by ecdysone. A demonstration of synthesis of the enzyme de novo.
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ABSTRACT:
SUBMITTER: Fragoulis EG
PROVIDER: S-EPMC1165281 | biostudies-other | 1975 Jan
REPOSITORIES: biostudies-other
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Project description:The study of DOPA (3,4-dihydroxyphenylalanine) decarboxylase by steady-state methods is difficult because multiple reactions occur. The reaction with DOPA was studied at enzyme concentrations between 20 and 50 micrometer by direct observation of the bound coenzyme by using stopped-flow and conventional spectrophotometry. Four processes were observed on different time scales and three of these were attributed to stages in the decarboxylation. The fourth was attributed to an accompanying transamination that renders the enzyme inactive. It was clear that much, if not all, of the 330 nm-absorbing coenzyme present in the free enzyme plays an active part in the decarboxylation, since it is converted into 420 nm-absorbing material in the first observable step. An intermediate absorbing maximally at 390 nm is formed in a slower step. Rate and equilibrium constants have been determined and the ratio of decarboxylation to transamination was estimated to be 1200:1.
| S-EPMC1161566 | biostudies-other
Project description:1. When homogenates of brains from mature adult locusts (Locusta migratoria) were incubated with l-3-(3,4-dihydroxyphenyl)[3-(14)C]alanine the major radioactive metabolite was dopamine, suggesting the presence of a dopa (3,4-dihydroxyphenylalanine) decarboxylase. 2. Decarboxylation of l-dopa by this tissue, measured under optimum conditions by a radiochemical method, was 21mumol of CO(2)/h per g wet wt. Apparent decarboxylation of l-tyrosine proceeded at 0.34mumol of CO(2)/h per g wet wt. There was no detectable decarboxylation of l-tryptophan, l-histidine or l-phenylalanine. 3. Dopa decarboxylase activity was found in all major regions of the ventral nerve cord of the mature locust (range: 4-7mumol of CO(2)/h per g wet wt.) but was low or absent in thoracic peripheral nerve. 4. Marked decarboxylation of l-dopa was found in homogenates of brains of four other species of insects, and in brain and ventral nerve cord, but not in the claw nerve, of the crayfish. 5. The activity of the locust brain enzyme may be slightly lower at the time of imaginal ecdysis than during the mature period. By contrast, the dopa decarboxylase that produces dopamine as an intermediate in cuticle biosynthesis is known to be high in activity at the time of ecdysis and low in activity during the intermoult stages.
| S-EPMC1177643 | biostudies-other
Project description:The effect of guanidinium chloride (GuCl) on enzyme activity, hydrodynamic volume, circular dichroism, and fluorescence of 3,4-dihydroxyphenylalanine (Dopa) decarboxylase from pig kidney (pkDDC) was studied under equilibrium conditions. Unfolding proceeds in at least three stages. The first transition, occurring between 0 and 1 M GuCl, gives rise to a dimeric inactive species which has lost pyridoxal 5'-phosphate (PLP), and has a high tendency to aggregate, but retains almost all of the native spectroscopic characteristics. The second equilibrium transition, between 1 and 2.2 M GuCl, involves dimer dissociation, with some loss of tertiary and secondary structure. Additionally, gross conformational changes at or near the PLP microenvironment were detected by fluorescence of NaBH4-reduced enzyme. The third step, presumably representing complete unfolding of pkDDC, appears to be complete at 4.5 M GuCl, as indicated by the lack of further substantial changes in any of the signals being studied. Attempts at refolding resulted in the findings that: (1) partial reactivation is observed only starting from enzyme denatured at concentrations below 1.5 M GuCl, and (2) starting from completely denatured protein, the refolding process is apparently reversible down to concentrations of approx. 2 M GuCl. Taken together, this would seem to indicate that the monomer-dimer transition is impaired under the experimental conditions tested. A plausible model is presented for the unfolding/refolding of pkDDC.
| S-EPMC1134907 | biostudies-other
Project description:Background3,4-Dihydroxyphenylalanine decarboxylase (DDC), also known as aromatic L-amino acid decarboxylase, catalyzes the decarboxylation of a number of aromatic L-amino acids. Physiologically, DDC is responsible for the production of dopamine and serotonin through the decarboxylation of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively. In insects, both dopamine and serotonin serve as classical neurotransmitters, neuromodulators, or neurohormones, and dopamine is also involved in insect cuticle formation, eggshell hardening, and immune responses.Principal findingsIn this study, we expressed a typical DDC enzyme from Drosophila melanogaster, critically analyzed its substrate specificity and biochemical properties, determined its crystal structure at 1.75 Angstrom resolution, and evaluated the roles residues T82 and H192 play in substrate binding and enzyme catalysis through site-directed mutagenesis of the enzyme. Our results establish that this DDC functions exclusively on the production of dopamine and serotonin, with no activity to tyrosine or tryptophan and catalyzes the formation of serotonin more efficiently than dopamine.ConclusionsThe crystal structure of Drosophila DDC and the site-directed mutagenesis study of the enzyme demonstrate that T82 is involved in substrate binding and that H192 is used not only for substrate interaction, but for cofactor binding of drDDC as well. Through comparative analysis, the results also provide insight into the structure-function relationship of other insect DDC-like proteins.
| S-EPMC2809104 | biostudies-literature
Project description:We report a facile approach to the synthesis of acetonide and Fmoc protected 3,4-dihydroxyphenylalanine (DOPA), Fmoc-DOPA(acetonide)-OH. By protecting the amino group of DOPA with a phthaloyl group and the carboxyl group as a methyl ester, acetonide protection of the catechol of DOPA derivative was realized in the presence of p-toluenesulfonic acid. Following removal of protecting groups, the intermediate was converted to Fmoc-DOPA(acetonide)-OH, which was successfully incorporated into a short DOPA-containing peptide, derived from marine tubeworm cement proteins Pc1 and Pc2.
| S-EPMC2598405 | biostudies-literature
Project description:A major product of hydroxy-radical addition to tyrosine is 3, 4-dihydroxyphenylalanine (DOPA) which has reducing properties. Protein-bound DOPA (PB-DOPA) has been shown to be a major component of the stable reducing species formed during protein oxidation under several conditions. The aim of the present work was to investigate whether DOPA, and especially PB-DOPA, can mediate oxidative damage to DNA. We chose to generate PB-DOPA using mushroom tyrosinase, which catalyses the hydroxylation of tyrosine residues in protein. This permitted us to study the reactions of PB-DOPA in the virtual absence of other protein-bound oxidation products. The formation of two oxidation products of DNA, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodG) and 5-hydroxy-2'-deoxycytidine (5OHdC), were studied with a novel HPLC using gradient elution and an electrochemical detection method, which allowed the detection of both DNA modifications in a single experiment. We found that exposure of calf thymus DNA to DOPA or PB-DOPA resulted in the formation of 8oxodG and 5OHdC, with the former predominating. The formation of these DNA oxidation products by either DOPA or PB-DOPA depended on the presence of oxygen, and also on the presence and on the concentration of transition metal ions, with copper being more effective than iron. The yields of 8oxodG and 5OHdC increased with DOPA concentration in proteins. Thus PB-DOPA was able to promote further radical-generating events, which then transferred damage to other biomolecules such as DNA.
| S-EPMC1219245 | biostudies-other
Project description:Detection of the drug Levodopa (3,4-dihydroxyphenylalanine, L-Dopa) is essential for the medical treatment of several neural disorders, including Parkinson's disease. In this paper, we employed surface-enhanced Raman scattering (SERS) with three shapes of silver nanoparticles (nanostars, AgNS; nanospheres, AgNP; and nanoplates, AgNPL) to detect L-Dopa in the nanoparticle dispersions. The sensitivity of the L-Dopa SERS signal depended on both nanoparticle shape and L-Dopa concentration. The adsorption mechanisms of L-Dopa on the nanoparticles inferred from a detailed analysis of the Raman spectra allowed us to determine the chemical groups involved. For instance, at concentrations below/equivalent to the limit found in human plasma (between 10-7-10-8 mol/L), L-Dopa adsorbs on AgNP through its ring, while at 10-5-10-6 mol/L adsorption is driven by the amino group. At even higher concentrations, above 10-4 mol/L, L-Dopa polymerization predominates. Therefore, our results show that adsorption depends on both the type of Ag nanoparticles (shape and chemical groups surrounding the Ag surface) and the L-Dopa concentration. The overall strategy based on SERS is a step forward to the design of nanostructures to detect analytes of clinical interest with high specificity and at varied concentration ranges.
| S-EPMC6982777 | biostudies-literature
Project description:The catechol group of 3,4-dihydroxyphenylalanine (L-DOPA) derived from L-tyrosine oxidation is a key post-translational modification (PTM) in many protein biomaterials and has potential as a bioorthogonal handle for precision protein conjugation applications such as antibody-drug conjugates. Despite this potential, indiscriminate enzymatic modification of exposed tyrosine residues or complete replacement of tyrosine using auxotrophic hosts remains the preferred method of introducing the catechol moiety into proteins, which precludes many protein engineering applications. We have developed new orthogonal translation machinery to site-specifically incorporate L-DOPA into recombinant proteins and a new fluorescent biosensor to selectively monitor L-DOPA incorporation in vivo. We show simultaneous biosynthesis and incorporation of L-DOPA and apply this translation machinery to engineer a novel metalloprotein containing a DOPA-Fe chromophore.
| S-EPMC8217333 | biostudies-literature
Project description:A series of phosphonic analogues of tyrosine and 3,4-dihydroxyphenylalanine (dopa) were synthesized in order to study their interaction with mushroom tyrosinase. 1-Amino-2-(3,4-dihydroxyphenyl)ethylphosphonic acid and 1-amino-2-(3,4-dimethoxyphenyl)ethylphosphonic acid turned out to be substrates for mushroom tyrosinase with Km values of 3.3 mM and 9.3 mM respectively. Shortening of the alkyl chain by one methylene group gave amino-(3,4-dihydroxyphenyl)methylphosphonic acid, one of the most powerful known inhibitors of this enzyme. This compound, racemic as well as in its optically active forms, exerts a mixed type of inhibition with an affinity for the enzyme one order of magnitude greater than that of the natural substrate.
| S-EPMC1147667 | biostudies-other
Project description:Silk composites with natural rubber (NR) were prepared by mixing degummed silk and NR latex solutions. A significant enhancement of the mechanical properties was confirmed for silk/NR composites compared to a NR-only product, indicating that silk can be applied as an effective reinforcement for rubber materials. Attenuated total reflection Fourier transform infrared (ATR-FTIR) and wide-angle X-ray diffraction (WAXD) analysis revealed that a β-sheet structure was formed in the NR matrix by increasing the silk content above 20 wt%. Then, 3,4-dihydroxyphenylalanine (DOPA)-modified silk was also blended with NR to give a DOPA-silk/NR composite, which showed superior mechanical properties to those of the unmodified silk-based composite. Not only the chemical structure but also the dominant secondary structure of silk in the composite was changed after DOPA modification. It was concluded that both the efficient adhesion property of DOPA residue and the secondary structure change improved the compatibility of silk and NR, resulting in the enhanced mechanical properties of the formed composite. The knowledge obtained herein should contribute to the development of the fabrication of novel silk-based elastic materials.
| S-EPMC6982767 | biostudies-literature