• Journal of Microbiology
• /
• 제35권4호
• /
• pp.300-308
• /
• 1997

The catechol 2,3-dioxygenase (C23O) encoded by the Pseudomonas putida xylE gene was over-produced in Escherichia coli and purified to homogeneity. The activity of the C23O required the reduced form of the Fe(II) ion since the enzyme was highly susceptible to inactivation with hydrogen perocide but reactivated with the addition of ferrous sulfate in conjunction with ascorbic acid. The C23O activity was abolished by treatment with the chemical reagents, diethyl-pyrocarbonate (DEPC), tetranitromethane (TNM), and 1-cyclohexy1-3-(2-morpholinoethyl) car-bodiimidemetho-ρ-toluenesulfontate (CMC), which are modifying reagents of histidine, tyrosine and glutamic acid, respectively. These results suggest that histidine, tyrosine and glutamic acid residues may be good active sites for the enzyme activity. These amino acid residues are conserved residues may be good active sites for the enzyme activity. These amino acid residues are conserved residues among several extradion dioxygenases and have the chemical potential to serveas ligands for Fe(II) coordination. Analysis of random point mutants in the C23O gene derived by PCR technique revealed that the mutated positions of two mutants, T179S and S211R, were located near the conserved His165 amd Hos217 residues, respectively. This finding indicates that these two positions, along with the conserved histidine residues, are specially effective regions for the enzyme function.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2003년도 생물공학의 동향(XII)
• /
• pp.422-427
• /
• 2003

Streptomyces setonii (ATCC 39116) is a Gram-positive thermophilic soil actinomycetes capable of degrading single aromatic compounds including phenol and benzoate via ortho-cleavage pathway. we isolated approximately 6.3-kb S. setonii DNA fragment containing a thermophilic catechol 1,2-dioxygenase(C12O) gene. Here we further revealed that the 6.3-kb S. setonii DNA fragment was organized into two putative divergently-transcribed clusters with 6 complete and one incomplete open reading frames (ORFs). The first cluster with 3 ORFs showed significant homologies to previously known benA, benB, and benC, implying a part of benzoate catabolic operon. The second cluster revealed an ortho-cleavage catechol catabolic operon with three translationally-coupled ORFs (catR, catB, catA). Each of these individually-cloned ORFs was expressed in E. coli and identified as a distinct protein band with a theoretical molecular weight in SDS-PAGE. The expression of the cloned S. setonii catechol operon was induced in a heterologous S. lividans by specific single aromatic compounds including catechol, phenol, and 4-chlorophenol. The simitar induction pattern was also observed using a luciferase gene-fused reporter system, implying that S. setonii employs an inducer-specific regulatory mechanism for aromatic compound metabolism.