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 degenerate oligonucleotides designed using known amino acid sequences. A 1.5-kb SmaI fragment from a 15-kb primary clone was subcloned, sequenced, and shown to contain two successive open reading frames, designated pcaH 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 positions. Cloning of pcaHG into broad-host-range expression vector pKMY319 allowed high levels of expression in P. putida strains, as well as in Proteus mirabilis after specific induction of the plasmid-encoded nahG promoter with salicylate. The recombinant enzyme was purified and crystallized from P. mirabilis, which lacks an endogenous 3,4-PCD. The physical, spectroscopic, and kinetic properties of the recombinant enzyme were indistinguishable from those of the wild-type enzyme. Moreover, the same transient enzyme intermediates were formed during the catalytic cycle. These studies establish the methodology which will allow mechanistic 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.
Frazee
R W
RW
Department of Biochemistry, Medical School, University of Minnesota, Minneapolis 55455.
Livingston
D M
DM
LaPorte
D C
DC
Lipscomb
J D
JD
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D005798:Genes, Bacterial; D015698:Genomic Library; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D005810:Multigene Family; D015345:Oligonucleotide Probes; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011513:Proteus mirabilis / Q000201:enzymology / Q000235:genetics; D011520:Protocatechuate-3,4-Dioxygenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013053:Spectrophotometry
D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D011520:Protocatechuate-3,4-Dioxygenase