RESUMO
The E. coli gene pdxH encoding pyridoxol-5'-phosphate:oxygen oxidoreductase (deaminating) (EC 1.4.3.5, PdxH) was cloned, located to phage 20B5 of the library of Kohara et al. (Kohara, Y, Akiyama, K. and Isono K. (1987) Cell 50, 495-508) and assigned to a stretch between 36.0 and 36.1 min of the E. coli chromosome. The gene was overexpressed as a MBP/PdxH fusion protein. The fusion protein was purified by affinity chromatography on an amylose resin and hydrolyzed in the presence of protease 'factor Xa' resulting in homogeneous PdxH protein after another column chromatography. Both the MBP/PdxH fusion protein and the PdxH protein were characterized. Both enzymes are FMN-dependent enzymes which oxidize pyridoxol phosphate and pyridoxamine phosphate in the presence of oxygen to pyridoxal phosphate. Km values of both proteins were similar ranging from 350 to 400 microM for the two substrates. The enzymes did not accept non-phosphorylated substrates. Kinetic data indicate that the enzyme (MBP/PdxH) is product inhibited (Ki 8 microM) by pyridoxal phosphate as a mixed type inhibitor. As revealed by gel exclusion chromatography a minor fraction of the fusion protein formed a dimer, whereas the bulk amount of protein was a monomer. No indication was found that the PdxH protein forms a dimer. The monomer was shown to be catalytically active.
Assuntos
Escherichia coli/enzimologia , Piridoxaminafosfato Oxidase/isolamento & purificação , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Genes Bacterianos , Cinética , Dados de Sequência Molecular , Fosfato de Piridoxal/biossíntese , Fosfato de Piridoxal/química , Piridoxaminafosfato Oxidase/antagonistas & inibidores , Piridoxaminafosfato Oxidase/genética , Piridoxina/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Mutants of Escherichia coli (pdx B and pdx C) which are blocked in the biosynthesis of pyridoxol (vitamin B6) showed a growth response to 4-hydroxy-L-threonine. This observation constitutes the first direct evidence in support of the view that 4-hydroxy-L-threonine is implicated in the biosynthesis of vitamin B6. 1-Aminopropan-2,3-diol, the decarboxylation product of 4-hydroxy-L-threonine, does not support the growth of these mutants. Deuterium from deuterium-labelled 1-aminopropan-2,3-diol was not incorporated into pyridoxol.