MOLECULAR-CLONING AND HOMOLOGY MODELING OF PROTOCATECHUATE 3,4-DIOXYGENASE FROM PSEUDOMONAS-MARGINATA

The genes that encode the alpha and beta subunits of protocatechuate 3 ,4-dioxygenase (3,4-PCD[EC 1.13.11.3]) were cloned from a Pseudomonas marginata genomic library. These genes pcaG and pcaH, were found when screening the library for hydrolase genes. The two open reading frames of the PCD genes could be identified adjacent to an esterase gene by sequence homology. A 1.7-kb KpnI/ApaI fragment, carrying pcaG and pcaH , was subcloned and the genes were functionally expressed in Escherich ia coli. The deduced amino acid sequence shows high homology to previo usly determined amino acid sequences of bacterial protocatechuate 3,4- dioxygenases. A homology model based on the available crystal structur e of the protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa s hows high similarity with the binding and catalytic sites.