Substitution, Insertion, Deletion, Suppression, and Altered Substrate Specificity in Functional Protocatechuate 3,4-Dioxygenases

Abstract

Protocatechuate 3,4-dioxygenase is a member of a family of bacterial enzymes that cleave the aromatic rings of their substrates between two adjacent hydroxyl groups, a key reaction in microbial metabolism of varied environmental chemicals. In an appropriate genetic background, it is possible to select for Acinetobacterstrains containing spontaneous mutations blocking expression ofpcaH or -G, genes encoding the α and β subunits of protocatechuate 3,4-dioxygenase. The crystal structure of the Acinetobacter oxygenase has been determined, and this knowledge affords us the opportunity to understand how mutations alter function in the enzyme. An earlier investigation had shown that a large fraction of spontaneous mutations inactivatingAcinetobacter protocatechuate oxygenase are either insertions or large deletions. Therefore, the prior procedure of mutant selection was modified to isolate Acinetobacter strains in which mutations within pcaH or -G cause a heat-sensitive phenotype. These mutations affected residues distributed throughout the linear amino acid sequences of PcaH and PcaG and impaired the dioxygenase to various degrees. Four of 16 mutants had insertions or deletions in the enzyme ranging in size from 1 to 10 amino acid residues, highlighting areas of the protein where large structural changes can be tolerated. To further understand how protein structure influences function, we isolated strains in which the phenotypes of three different deletion mutations in pcaH or -G were suppressed either by a spontaneous mutation or by a PCR-generated random mutation introduced into theAcinetobacter chromosome by natural transformation. The latter procedure was also used to identify a single amino acid substitution in PcaG that conferred activity towards catechol sufficient for growth with benzoate in a strain in which catechol 1,2-dioxygenase was inactivated.

Spontaneous mutations affecting transcriptional regulation by protocatechuate in Acinetobacter

“…In PobR, a cold-sensitive mutation producing a Trp 80 to Arg 80 substitution near the helix-turn-helix motif generated a protein that bound abnormally tightly to its operator DNA [1]. Another conditional pcaU mutation (also with a stronger phenotype at the restrictive temperature than the nine leaky mutations apparently inactivating PcaU) was identi¢ed in this study: in a previously isolated strain [10], a Gly 150 to Arg 150 substitution conferred heatsensitive growth with protocatechuate (Table 1).…”

“…This block prevented the toxic accumulation of carboxymuconate due to the vpcaBDK1 mutation ( Fig. 1) preferentially during growth at 37³C relative to growth at 22³C [10].…”

“…Single colonies of succinate-grown ADP500 [9] were transferred to patches on agar with 10 mM succinate and 3 mM protocatechuate incubated at 22³C. Derivatives of ADP500 in which a spontaneous mutation blocked protocatechuate catabolism were isolated as described previously [10] but with the following exceptions: only the smallest colonies of cells resistant to protocatechuate were chosen (after incubation for 2^6 days); of these strains, those with better growth in the presence of p-hydroxybenzoate at 22³C than at 37³C (20% of those tested, on average) were saved and puri¢ed on Luria^Bertani agar ; after correction of vpcaBDK1, the spontaneous mutation was localized to regulatory elements of protocatechuate catabolism (and not to the structural pcaH and -G genes) by marker rescue using plasmid pZR15 [4]; and template DNA for sequencing was ampli¢ed using primers I8PCAU1 (5P-CTATTGAGGTTACACGTAGATCG-3P) and I8PCAU4 (5P-GATGAATCAGATCGATATGGCA-A-3P).…”

“…An earlier study [10] described a procedure to e¤ciently isolate strains with a spontaneous heat-sensitive mutation in the genes for protocatechuate dioxygenase [12]. Such strains were identi¢ed by their heat-enhanced block in protocatechuate catabolism.…”

“…1). Such selection yielded strains with spontaneous loss-of-function mutations in the structural gene pobA [6,7] or the regulatory gene pobR [2,8], or in the structural genes pcaH and pcaG [9,10]. Nearly completely missing, however, were regulatory mutations a¡ecting protocatechuate catabolism : only the pcaP1 promoter mutation has been described to date [9].…”