Purification, characterization and gene cloning of 6-hydroxynicotinate 3-monooxygenase from Pseudomonas fluorescens TN5.
6-Hydroxynicotinate 3-monooxygenase, a membrane-bound, 42-kDa monomeric enzyme from Pseudomonas fluorescens TN5 was purified and characterized. The enzyme catalyzes the oxidative decarboxylation of 6-hydroxynicotinate and depends on O2, NADH and FAD with the holoenzyme containing 1 M of FAD per 1 M of enzyme. The isolated enzyme was used for the synthesis of 2,5-dihydroxypyridine, a precursor for the chemical synthesis of 5-aminolevulinic acid, which is applied as a plant growth hormone, a herbicide and in cancer therapy. A 1.8-kbp DNA fragment, which contains the ORF encoding 6-hydroxynicotinic acid 3-monooxygenase, was cloned, sequenced and expressed in Escherichia coli. The deduced 385 amino acid sequence of the cloned ORF is in agreement with the enzyme molecular mass, amino acid sequence of an internal peptide, contains a putative FAD-binding site and is homologous to similar flavoproteins such as salicylate 1-monoxygenase.
Nakano
H
H
Department of Food Science and Technology, Nagoya University, Japan.
Wieser
M
M
Hurh
B
B
Kawai
T
T
Yoshida
T
T
Yamane
T
T
Nagasawa
T
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004790:Enzyme Induction / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005420:Flavoproteins / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics; D011725:Pyridines / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D005420:Flavoproteins; D011725:Pyridines; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C118332:6-hydroxynicotinate 3-monooxygenase; C022619:salicylate 1-monooxygenase; C065452:2,5-dihydroxypyridine