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.
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-pyridinedicarboxylic acid. Crude extracts of strain 10d grown on 4-amino-3-hydroxybenzoic acid converted 2-amino-5-carboxymuconic 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 (epsilonmax = 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-hydroxybenzoic 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-hydroxymuconic 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-carboxymuconic 6-semialdehyde. The deamination step in the proposed pathway differs from that in the pathways for 2-aminophenol and its derivatives.
Orii
Chika
C
Division of Science of Biological Resources, Graduate School of Science and Technology, Kobe University, Rokko, Kobe, Japan.
Takenaka
Shinji
S
Murakami
Shuichiro
S
Aoki
Kenji
K
D010129:4-Aminobenzoic Acid / Q000737:chemistry / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism; D000619:Aminohydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001884:Bordetella / Q000145:classification* / Q000201:enzymology* / Q000378:metabolism; D002457:Cell Extracts / Q000302:isolation & purification; D003641:Deamination; D062385:Hydroxybenzoates / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D013057:Spectrum Analysis; D062366:para-Aminobenzoates
C489259:4-amino-3-hydroxybenzoic acid; D000447:Aldehydes; D002457:Cell Extracts; D062385:Hydroxybenzoates; D062366:para-Aminobenzoates; C489260:2-amino-5-carboxymuconic 6-semialdehyde deaminase; D000619:Aminohydrolases; D010129:4-Aminobenzoic Acid