The aniline-assimilating bacterium Rhodococcus sp. AN-22 was found to constitutively synthesize CatB (cis,cis-muconate cycloisomerase) and CatC (muconolactone isomerase) in its cells growing on non-aromatic substrates, in addition to the previously reported CatA (catechol 1,2-dioxygenase). The bacterium maintained the specific activity of the three enzymes at an almost equal level during cultivation on succinate. CatB and CatC were purified to homogeneity and characterized. CatB was a monomer with a molecular mass of 44 kDa. The enzyme was activated by Mn2+, Co2+ and Mg2+. Native CatC was a homo-octamer with a molecular mass of 100 kDa. The enzyme was stable between pH 7.0 and 10.5 and was resistant to heating up to 90 °C. Genes coding for CatA, CatB and CatC were cloned and named catA, catB and catC respectively. The catABC genes were transcribed as one operon. The deduced amino acid sequences of CatA, CatB and CatC showed high identities with those from other Gram-positive micro-organisms. A regulator gene such as catR encoding a regulatory protein was not observed around the cat gene cluster of Rhodococcus sp. AN-22, but a possible relic of catR was found in the upstream region of catA. Reverse transcriptase-PCR and primer extension analyses showed that the transcriptional start site of the cat gene cluster was located 891 bp upstream of the catA initiation codon in the AN-22 strain growing on both aniline and succinate. Based on these data, we concluded that the bacterium constitutively transcribed the catABC genes and translated its mRNA into CatA, CatB and CatC.
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Research Article|
December 12 2005
Constitutive expression of catABC genes in the aniline-assimilating bacterium Rhodococcus species AN-22: production, purification, characterization and gene analysis of CatA, CatB and CatC
Eitaro Matsumura;
Eitaro Matsumura
*Division of Life Science, Graduate School of Science and Technology, Kobe University, Rokko, Kobe 657-8501, Japan
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Masashi Sakai;
Masashi Sakai
†Laboratory of Applied Microbiology, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Rokko, Kobe 657-8501, Japan
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Katsuaki Hayashi;
Katsuaki Hayashi
†Laboratory of Applied Microbiology, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Rokko, Kobe 657-8501, Japan
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Shuichiro Murakami;
Shuichiro Murakami
†Laboratory of Applied Microbiology, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Rokko, Kobe 657-8501, Japan
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Shinji Takenaka;
Shinji Takenaka
†Laboratory of Applied Microbiology, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Rokko, Kobe 657-8501, Japan
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Kenji Aoki
Kenji Aoki
1
†Laboratory of Applied Microbiology, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Rokko, Kobe 657-8501, Japan
1To whom correspondence should be addressed (email kaoki@kobe-u.ac.jp).
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Publisher: Portland Press Ltd
Received:
May 04 2005
Revision Received:
August 30 2005
Accepted:
September 13 2005
Accepted Manuscript online:
September 13 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 393 (1): 219–226.
Article history
Received:
May 04 2005
Revision Received:
August 30 2005
Accepted:
September 13 2005
Accepted Manuscript online:
September 13 2005
Citation
Eitaro Matsumura, Masashi Sakai, Katsuaki Hayashi, Shuichiro Murakami, Shinji Takenaka, Kenji Aoki; Constitutive expression of catABC genes in the aniline-assimilating bacterium Rhodococcus species AN-22: production, purification, characterization and gene analysis of CatA, CatB and CatC. Biochem J 1 January 2006; 393 (1): 219–226. doi: https://doi.org/10.1042/BJ20050740
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