Protocatechuate 3,4-dioxygenase plays a central role in the microbial removal of plant materials from the environment. The enzyme has been crystallized, and biochemical analysis has elucidated key amino acid residues which activate oxygen for intradiol cleavage. Yet to be determined are more subtle roles played by additional amino acid residues and the extent to which conserved amino acid residues are tolerant of substitution in a functional enzyme. Such residues will be targeted by a novel genetic procedure that combines Polymerase Chain Reaction (PCR), random mutagenesis, natural transformation and selection for loss-of-function to obtain Acinetobacter mutants in which substitutions impairing protocatechuate 3,4-dioxygenase function can be identified at the level of phenotype (null, leaky or temperature-conditional). A previously isolated Acinetobacter mutant acquired low level catechol 1,2-dioxygenase activity as it lost protocatechuate 3,4-dioxygenase activity. PCR-mutagenesis will be used to improve the catechol 1,2-dioxygenase activity of the mutant enzyme and to test the hypothesis that an enzyme that acts effectively upon both catechol and protocatechuate can be selected. The alpha (PcaG) and beta (PcaH) subunits of protocatechuate 3,4-dioxygenase share common ancestry, and similar peptides within the protein subunits are required to maintain a symmetrical conformation in the alphabetaFe3+ protomer. Thus selection at the level of protein demands nucleotide sequence repetitions in pcaH and pcaG, the neighboring structural genes for the enzyme. Mispairing between complementary strands of repeated sequences in DNA can guide deletion mutations. Such a genetic hazard appears to have been largely overcome in Acinetobacter pcaHG because the deletions that do occur in these genes do not appear to be guided by the conserved nucleotide sequence repetitions. Within pcaH, and flanking a nucleotide sequence conserved with pcaG, is a hotspot for short duplications caused by localized strand slippage. This finding fosters the hypothesis that localized DNA strand slippage may deter recombinational events causing deletions guided by nucleotide sequences shared by pcaH and pcaG. The hypothesis will be tested as will hypotheses predicting the effect of replication and temperature on localized DNA strand slippage, the influence of sequence similarity on deletion formation, and the effect of distance between repeated sequences on the frequency of deletions. Throughout the proposed research, specific selection procedures will target recovery of relevant mutants with the exclusion of others. Thus a unified technology will be applied to defining factors that determine variability and constancy in both protocatechuate 3,4-dioxygenase and its structural genes.
