Gene cloning and characterization of a deaminase from the 4-amino-3-hydroxybenzoate-assimilating Bordetella sp. strain 10d.
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.
Takenaka
Shinji
S
Department of Applied Biological Chemistry, Graduate School of Agricultural Science, Kobe University, Kobe, Japan. hakko3@kobe-u.ac.jp
Sato
Tomomi
T
Koshiya
Jyun
J
Murakami
Shuichiro
S
Aoki
Kenji
K
D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001884:Bordetella / Q000201:enzymology*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005231:Fatty Acids, Unsaturated; D011994:Recombinant Proteins; C002091:hydroxymuconic semialdehyde; C489260:2-amino-5-carboxymuconic 6-semialdehyde deaminase; D000619:Aminohydrolases