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Molecular cloning and sequencing of a cDNA of rat dopa decarboxylase: partial amino acid homologies with other enzymes synthesizing catecholamines.

ABSTRACT: Dopa decarboxylase (DDC; aromatic-L-amino-acid decarboxylase; aromatic-L-amino-acid carboxylase, EC 4.1.1.28) was purified from rat liver and its partial sequence was determined. Synthetic oligonucleotides were used to construct and screen rat liver cDNA libraries, and three clones were isolated and sequenced. The 2 kilobases of DDC cDNA cloned consisted of a 5'-noncoding segment of 78 nucleotides, a coding region of 1440 nucleotides, and a 3'-noncoding region of 438 nucleotides. The encoded protein of 480 amino acid residues had a molecular weight of 54,000. A special feature of the primary structure of rat DDC was a repeating structure consisting of 29 amino acid residues. A sequence of 58 amino acid residues, including this repeating structure of rat DDC, was found to show homologies with those of rat tyrosine hydroxylase, human dopamine beta-hydroxylase, and bovine phenylethanolamine N-methyltransferase, other mammalian enzymes that synthesize catecholamines. These results indicate that catecholamine biosynthetic enzymes are structurally related and suggest that their homologous domains are important for catechol-protein interactions.

SUBMITTER: Tanaka T

PROVIDER: S-EPMC298231 | biostudies-other | 1989 Oct

REPOSITORIES: biostudies-other

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Molecular cloning and sequencing of a cDNA of rat dopa decarboxylase: partial amino acid homologies with other enzymes synthesizing catecholamines.

Tanaka T T Horio Y Y Taketoshi M M Imamura I I Ando-Yamamoto M M Kangawa K K Matsuo H H Kuroda M M Wada H H

Proceedings of the National Academy of Sciences of the United States of America 19891001 20

Dopa decarboxylase (DDC; aromatic-L-amino-acid decarboxylase; aromatic-L-amino-acid carboxylase, EC 4.1.1.28) was purified from rat liver and its partial sequence was determined. Synthetic oligonucleotides were used to construct and screen rat liver cDNA libraries, and three clones were isolated and sequenced. The 2 kilobases of DDC cDNA cloned consisted of a 5'-noncoding segment of 78 nucleotides, a coding region of 1440 nucleotides, and a 3'-noncoding region of 438 nucleotides. The encoded pro ...[more]

PMID: 2813383

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Project description: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.

Project description:BACKGROUND: Dopa decarboxylase (DDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyzes the decarboxylation of L-Dopa to dopamine, and involved in complex neuroendocrine-immune regulatory network. The function for DDC in the immunomodulation remains unclear in invertebrate. METHODOLOGY: The full-length cDNA encoding DDC (designated CfDDC) was cloned from mollusc scallop Chlamys farreri. It contained an open reading frame encoding a polypeptide of 560 amino acids. The CfDDC mRNA transcripts could be detected in all the tested tissues, including the immune tissues haemocytes and hepatopancreas. After scallops were treated with LPS stimulation, the mRNA expression level of CfDDC in haemocytes increased significantly (5.5-fold, P<0.05) at 3 h and reached the peak at 12 h (9.8-fold, P<0.05), and then recovered to the baseline level. The recombinant protein of CfDDC (rCfDDC) was expressed in Escherichia coli BL21 (DE3)-Transetta, and 1 mg rCfDDC could catalyze the production of 1.651±0.22 ng dopamine within 1 h in vitro. When the haemocytes were incubated with rCfDDC-coated agarose beads, the haemocyte encapsulation to the beads was increased significantly from 70% at 6 h to 93% at 24 h in vitro in comparison with that in the control (23% at 6 h to 25% at 24 h), and the increased haemocyte encapsulation was repressed by the addition of rCfDDC antibody (which is acquired via immunization 6-week old rats with rCfDDC). After the injection of DDC inhibitor methyldopa, the ROS level in haemocytes of scallops was decreased significantly to 0.41-fold (P<0.05) of blank group at 12 h and 0.47-fold (P<0.05) at 24 h, respectively. CONCLUSIONS: These results collectively suggested that CfDDC, as a homologue of DDC in scallop, modulated the immune responses such as haemocytes encapsulation as well as the ROS level through its catalytic activity, functioning as an indispensable immunomodulating enzyme in the neuroendocrine-immune regulatory network of mollusc.

Dataset Information

Molecular cloning and sequencing of a cDNA of rat dopa decarboxylase: partial amino acid homologies with other enzymes synthesizing catecholamines.

Publications

Molecular cloning and sequencing of a cDNA of rat dopa decarboxylase: partial amino acid homologies with other enzymes synthesizing catecholamines.

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