Molecular characterization of the genes pcaG and pcaH, encoding protocatechuate 3,4-dioxygenase, which are essential for vanillin catabolism in Pseudomonas sp. strain HR199

Pseudomonas sp, strain HR199 is able to utilize eugenol (4-allyl-2-methoxyp henol), vanillin (4-hydroxy-3-methoxybenzaldehyde), or protocatechuate as t he sole carbon source for growth. Mutants of this strain which were impaire d in the catabolism of vanillin but retained the ability to utilize eugenol or protocatechuate were obtained after nitrosoguanidine mutagenesis, One m utant (SK6169) was used as recipient of a Pseudomonas sp, strain HR199 geno mic library in cosmid pVK100, and phenotypic complementation was achieved w ith a 5.8-kbp EcoRI fragment (E58), The amino acid sequences deduced from t wo corresponding open reading frames (ORF) identified on E58 revealed high degrees of homology to pcaG and pcaH, encoding the two subunits of protocat echuate 3,4-dioxygenase. Three additional ORF most probably encoded a 4-hyd roxybenzoate 3-hydroxylase (PobA) and two putative regulatory proteins, whi ch exhibited homology to PcaQ of Agrobacterium tumefaciens and PobR of Pseu domonas aeruginosa, respectively. Since mutant SK6169 was also complemented by a subfragment of E58 that harbored only pcaH, this mutant was most prob ably lacking a functional beta subunit of the protocatechuate 3,4-dioxygena se. Since this mutant was still able to grow on protocatechuate and lacked protocatechuate 4,5-dioxygenase and protocatechuate 2,3-dioxygenase, the de gradation had to be catalyzed by different enzymes. Two other mutants (SK61 84 and SK6190), which were also impaired in the catabolism of vanillin, wer e not complemented by fragment E58, Since these mutants accumulated 3-carbo xy muconolactone during cultivation on eugenol, they most probably exhibite d a defect in a step of the catabolic pathway following the ortho cleavage. Moreover, in these mutants cyclization of 3-carboxymuconic acid seems to o ccur by a syn absolute stereochemical course, which is normally only observ ed for cis,cis-muconate lactonization in pseudomonads. In conclusion, vanil lin is degraded through the ortho-cleavage pathway in Pseudomonas sp, strai n HR199 whereas protocatechuate could also be metabolized via a different p athway in the mutants.