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

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