CLONING, SEQUENCING, AND EXPRESSION OF THE PSEUDOMONAS-PUTIDA PROTOCATECHUATE 3,4-DIOXYGENASE GENES

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 putida (formerly P. aeruginosa) (ATCC 23975) genomic library prepared in lambda phage. Plaques were screened by hybridization with degenera te oligonucleotides designed using known amino acid sequences. A 1.5-k b SmaI fragment from a 15-kb primary clone was subcloned, sequenced, a nd shown to contain two successive open reading frames, designated pca H and pcaG, corresponding to the beta and alpha subunits, respectively , of 3,4-PCD. The amino acid sequences deduced from pcaHG matched the chemically determined sequence of 3,4-PCD in all except three position s. Cloning of pcaHG into broad-host-range expression vector pKMY319 al lowed high levels of expression in P. putida strains, as well as in Pr oteus mirabilis after specific induction of the plasmid-encoded nahG p romoter with salicylate. The recombinant enzyme was purified and cryst allized from P. mirabilis, which lacks an endogenous 3,4-PCD. The phys ical, spectroscopic, and kinetic properties of the recombinant enzyme were indistinguishable from those of the wild-type enzyme. Moreover, t he same transient enzyme intermediates were formed during the catalyti c cycle. These studies establish the methodology which will allow mech anistic investigations to be pursued through site-directed mutagenesis of P. putida 3,4-PCD, the only aromatic ring-cleaving dioxygenase for which the three-dimensional structure is known.