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.