Purification, molecular cloning, and characterization of pyridoxine 4-oxidase from Microbacterium luteolum.
Pyridoxine 4-oxidase (EC 1.1.3.12, PN 4-oxidase), which catalyzes the oxidation of PN by oxygen or other hydrogen acceptors to form pyridoxal and hydrogen peroxide or reduced forms of the acceptors, respectively, was purified for the first time to homogeneity from Microbacterium luteolum YK-1 (=Aureobacterium luteolum YK-1). The purified enzyme required FAD for its catalytic activity and stability. The enzyme was a monomeric protein with the subunit molecular mass of 53,000 +/- 1,000 Da. PN was the only substrate as the hydrogen donor. Oxygen, 2,6-dichloroindophenol, and vitamin K3 were good substrates as the hydrogen acceptor. The gene (pno) encoding PN 4-oxidase was cloned. The gene encodes a protein of 507 amino acid residues corresponding to the molecular mass of the subunit. PN 4-oxidase was expressed in Escherichia coli and found to have the same properties as the native enzyme from M. luteolum YK-1. Comparisons of primary and secondary structures with other proteins showed that the enzyme belongs to the GMC oxidoreductase family. M. luteolum YK-1 has four plasmids. The pno gene was found on a chromosomal DNA. Search for genes similar in sequence in other organisms suggested that a nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, which harbors two plasmids, has a PN degradation pathway I in chromosomal DNA.
Kaneda
Yasuo
Y
Department of Bioresources Science, Faculty of Agriculture, Kochi University, Japan.
Ohnishi
Kouhei
K
Yagi
Toshiharu
T
D000192:Actinomycetales / Q000201:enzymology*; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases