PURIFICATION AND CHARACTERIZATION OF THE PYRIDOXOL-5'-PHOSPHATE-OXYGEN OXIDOREDUCTASE (DEAMINATING) FROM ESCHERICHIA-COLI

The E. call gene pdxH encoding pyridoxol-5'-phosphate:oxygen oxidoredu ctase (deaminating) (EC 1.4.3.5, PdxH) was cloned, located to phage 20 B5 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 M BP/PdxH fusion protein. The fusion protein was purified by affinity ch romatography on an amylose resin and hydrolyzed in the presence of pro tease 'factor Xa' resulting in homogeneous PdxH protein after another column chromatography. Both the MBP/PdxH fusion protein and the PdxH p rotein were characterized. Both enzymes are FMN-dependent enzymes whic h oxidize pyridoxol phosphate and pyridoxamine phosphate in the presen ce of oxygen to pyridoxal phosphate. K-m values of both proteins were similar ranging from 350 to 400 mu M for the two substrates. The enzym es did not accept non-phosphorylated substrates. Kinetic data indicate that the enzyme (MBP/PdxH) is product inhibited (K-i 8 mu M) by pyrid oxal 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 f ound that the PdxH protein forms a dimer. The monomer was shown to be catalytically active.