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 pyr idoxal 5'-phosphate (PLP)-dependent homodimeric enzyme that catalyses the decarboxylation of L-dopa and other L-aromatic amino acids. To adv ance 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 unexpectedl y observed that it possesses certain differences from the enzyme purif ied from pig kidney. Whereas the latter protein binds 1 molecule of PL P per dimer, the recombinant enzyme was found to bind two molecules of coenzyme per dimer. Moreover, the V-max was twice that of the protein purified from tissue. On addition of substrate, the absorbance change s accompanying transaldimination were likewise 2-fold greater in the r ecombinant enzyme. Examination of the respective apoenzymes by absorba nce, CD and fluorescence spectroscopy revealed distinct differences. T he recombinant apoprotein has no significant absorbance at 335 nm, unl ike the pig kidney apoenzyme; in the latter case this residual absorba nce 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 b road emission at about 400 nm. However, the holoenzyme-apoenzyme trans ition did not markedly alter the respective fluorescence properties of either recombinant or pig kidney DDC when excited at 335 nm. Taken to gether, these findings indicate that recombinant pig kidney DDC has tw o active-site PLP molecules and therefore displays structural characte ristics typical of PLP-dependent homodimeric enzymes. The natural enzy me contains one active-site PLP molecule whereas the remaining PLP bin ding site is most probably occupied by an inactive covalently bound co enzyme derivative; some speculations are made about its origin. The co enzyme absorbing bands of recombinant DDC show a modest pH dependence at 335 and 425 nm. A putative working model is presented to explain th is behaviour.