Cloning and expression of thermophilic catechol 1,2-dioxygenase gene (catA) from Streptomyces setonii.
Streptomyces setonii (ATCC 39116) degrades various single aromatic compounds such as phenol or benzoate via an ortho-cleavage pathway using catechol 1,2-dioxygenase (C12O). A PCR using degenerate primers based on the conserved regions of known C12O-encoding genes amplified a 0.45-kbp DNA fragment from S. setonii total DNA. A Southern hybridization analysis and size-selected DNA library screening using the 0.45-kbp PCR product as a probe led to the isolation of a 6.4-kbp S. setonii DNA fragment, from which the C12O-encoding genetic locus was found to be located within a 1.4-kbp DNA fragment. A complete nucleotide sequencing analysis of the 1.4-kbp DNA fragment revealed a 0.84-kbp open reading frame, which showed a strong overall amino acid similarity to the known high-G+C Gram-positive (but significantly less to the Gram-negative) bacterial mesophilic C12Os. The heterologous expression of the cloned 1.4-kbp DNA fragment in Escherichia coli demonstrated that this C12O possessed a thermophilic activity within a broad temperature range (up to 65 degrees C) and showed a higher activity against 3-methylcatechol than catechol or 4-methylcatechol, but no activity against protocatechuate.
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Department of Environmental Science, Hankuk University of Foreign Studies, 449-791, Kyunggi-Do, South Korea.
Park
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Kim
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D000595:Amino Acid Sequence; D001483:Base Sequence; D050548:Catechol 1,2-Dioxygenase; D003001:Cloning, Molecular; D049308:Dioxygenases; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013696:Temperature
D010105:Oxygenases; D049308:Dioxygenases; D050548:Catechol 1,2-Dioxygenase