Gene cloning and characterization of a deaminase from the 4-amino-3-hydroxybenzoate-assimilatingBordetellasp. strain 10d

Abstract

The 4-amino-3-hydroxybenzoate-assimilating Bordetella sp. strain 10d produces a deaminase that catalyzes the deamination of 2-amino-5-carboxymuconic 6-semialdehyde. A gene encoding the deaminase, ahdB, was cloned and expressed in Escherichia coli; ahdB is located downstream from the previously reported genes encoding 4-amino-3-hydroxybenzoate 2,3-dioxygenase (ahdA) and a LysR-type regulator. The deduced amino acid sequence of ahdB shows 30-33% identity to those of previously reported 2-aminomuconate deaminases. We identified a region (RAGDFLXVSG) conserved in AhdB and three other deaminases. The recombinant enzyme AhdB was purified to homogeneity. After a coupled enzyme assay with purified AhdA, AhdB, and the substrate 4-amino-3-hydroxybenzoate, the final product, formed by the action of AhdA, AhdB, and by nonenzymatic decarboxylation, was identified by HPLC, MS, and (1)H-nuclear magnetic resonance analyses as 2-hydroxymuconic 6-semialdehyde.

Comparative Genomic Analysis of the Regulation of Aromatic Metabolism in Betaproteobacteria

“…Proteins of this family quench reactive enamine/imine intermediates that can inactivate a number of enzymes by covalent binding to their active sites (Lambrecht et al, 2012; Flynn et al, 2013; Flynn and Downs, 2013). In particular, some YjgF-like proteins are known to deaminate short-lived enamines 2-aminomuconate to 4-oxalocrotonate during the degradation of nitrobenzene (He and Spain, 1998; Park and Kim, 2000) and intermediate 2-amino-5-carboxymuconic 6-semialdehyde into 2-hydroxy-5-carboxymuconic 6-semialdehyde in the meta -cleavage pathway of 4-amino-3-hydroxybenzoate degradation (Orii et al, 2004; Takenaka et al, 2009). Moreover, chorismatase XanB2 that converts chorismate into 3-hydroxybenzoate and 4-hydroxybenzoate has an YjgF-like domain and is important for the oxidative stress defense (Zhou et al, 2013).…”

Conserved YjgF Protein Family Deaminates Reactive Enamine/Imine Intermediates of Pyridoxal 5′-Phosphate (PLP)-dependent Enzyme Reactions

“…Finally, in Bordetella sp. strain 10d, AhdB deaminates 2-amino-5-carboxymuconic 6-semialdehyde, an unstable enamine intermediate (38,39). Interestingly, rutC in E. coli is included in a pyrimidine degradation operon, encoding a pathway that proceeds via the unstable enamine intermediate 3-aminoacrylate (40).…”

Bacterial degradation of monocyclic aromatic amines

“…The enzymes, 2-amino-3-hydroxybenzoate-2,3-dioxygenase and 2-amino-5-carboxymuconic-6-semialdehyde deaminase have been cloned and characterized from Bordetella sp. 10d ( Murakami et al, 2004 ; Takenaka et al, 2009 ).…”