TAILIEUCHUNG - Báo cáo khoa học: A novel coupled enzyme assay reveals an enzyme responsible for the deamination of a chemically unstable intermediate in the metabolic pathway of 4-amino-3-hydroxybenzoic acid inBordetellasp. strain 10d

2-Amino-5-carboxymuconic 6-semialdehyde is an unstable intermediate in the meta-cleavage pathway of 4-amino-3-hydroxybenzoic acid inBordetellasp. strain vitro, this compound is nonenzymatically converted to 2,5-pyrid-inedicarboxylic acid. Crude extracts of strain 10d grown on 4-amino-3-hydroxybenzoic acid converted 2-amino-5-car-boxymuconic 6-semialdehyde formed from 4-amino-3-hydroxybenzoic acid by the first enzyme in the pathway, 4-amino-3-hydroxybenzoate 2,3-dioxygenase, to a yellow compound (emax¼375 nm). The enzyme in the crude ex-tract carrying out the next stepwas purified to homogeneity | Eur. J. Biochem. 271 3248-3254 2004 FEBS 2004 doi A novel coupled enzyme assay reveals an enzyme responsible for the deamination of a chemically unstable intermediate in the metabolic pathway of 4-amino-3-hydroxybenzoic acid in Bordetella sp. strain 10d Chika Orii1 Shinji Takenaka2 Shuichiro Murakami2 and Kenji Aoki2 division of Science of Biological Resources Graduate School of Science and Technology department of Biofunctional Chemistry Faculty of Agriculture Kobe University Rokko Kobe Japan 2- Amino-5-carboxymuconic 6-semialdehyde is an unstable intermediate in the meta-cleavage pathway of 4-amino- 3- hydroxybenzoic acid in Bordetella sp. strain 10d. In vitro this compound is nonenzymatically converted to 2 5-pyrid-inedicarboxylic acid. Crude extracts of strain 10d grown on 4- amino-3-hydroxybenzoic acid converted 2-amino-5-car-boxymuconic 6-semialdehyde formed from 4-amino-3-hydroxybenzoic acid by the first enzyme in the pathway 4-amino-3-hydroxybenzoate 2 3-dioxygenase to a yellow compound emax 375 nm . The enzyme in the crude extract carrying out the next step was purified to homogeneity. The yellow compound formed from 4-amino-3-hydroxy-benzoic acid by this purified enzyme and purified 4-amino-3-hydroxybenzoate 2 3-dioxygenase in a coupled assay was identified as 2-hydroxymuconic 6-semialdehyde by GC-MS analysis. A mechanism for the formation of 2-hydroxy- muconic 6-semialdehyde via enzymatic deamination and nonenzymatic decarboxylation is proposed based on results of spectrophotometric analyses. The purified enzyme designated 2-amino-5-carboxymuconic 6-semialdehyde deaminase is a new type of deaminase that differs from the 2-aminomuconate deaminases reported previously in that it primarily and specifically attacks 2-amino-5-carboxymu-conic 6-semialdehyde. The deamination step in the proposed pathway differs from that in the pathways for 2-amino-phenol and its derivatives. Keywords 4-amino-3-hydroxybenzoic acid Bordetella sp.

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