UBC Theses and Dissertations
Protein purification and genetic characterization of a streptomycete protocatechuate 3,4-dioxygenase Iwagami, Sakura Grace
A previously uncharacterized Streptomyces sp. isolate 2065 was found to degrade vanillic acid and jc-hydroxybenzoic acid, utilizing these compounds as a sole carbon source. Induction of protocatechuate 3,4-dioxygenase (3,4-PCD [EC 18.104.22.168]), a ring-cleavage dioxygenase, when the strain was grown in the presence of vanillic acid or /j>-hydroxybenzoic acid was observed, indicating that this streptomycete isolate catabolizes both these lignin model compounds through the protocatechuic acid branch of the β-ketoadipate pathway. The 3,4-PCD was purified from cells grown in the presence of p-hydroxybenzoic acid. Two proteins, the α- and β -heterologous subunits of 3,4-PCD, were observed in approximately equimolar amounts on denaturing SDSPAGE. The α and β-subunits were found to correlate to ring fission activity. N-terminal protein sequence information was obtained, from which DNA oligonucleotides were designed and used in amplification of an 800-bp PCR product from isolate 2065 genomic DNA . The protein sequence of this 800-bp DNA fragment was found to be similar to the amino acid sequence for PcaH, the β subunit for 3,4-PCD. A bacteriophage X genomic library of 2065 was constructed and screened using the PCR product as a streptomycete pcaH gene probe; a 4.5-kb DNA fragment containing the structural genes for the α- and β- subunits of a protocatechuate 3,4- dioxygenase was cloned and the pcaG and pcaH genes were sequenced. The pcaG and pcaH genes encode 201 and 257 amino acid polypeptides with predicted sizes of 21,763 Da and 29,262 Da, respectively. The pcaGH genes and their predicted protein sequences were found to be similar to those from Burkholderia cepacia, Rhodococcus opacus, Pseudomonas putida, and Acinetobacter calcoaceticus.
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