Microbial metabolism of the pyridine ring. The hydroxylation of 4-hydroxypyridine to pyridine-3,4-diol (3,4-dihydroxypyridine) by 4-hydroxypyridine-3-hydroxylase.
1. The first metabolic step in the biodegradation of 4-hydroxypyridine by an Agrobacterium sp. was hydroxylation to form pyridine-3,4-diol. 2. Extracts required 1mol of O(2) and 1mol of NADH or NADPH for the conversion of 4-hydroxypyridine into pyridine-3,4-diol, suggesting that the enzyme responsible, 4-hydroxypyridine-3-hydroxylase, was a mixed function mono-oxygenase. 3. After treatment with acidic (NH(4))(2)SO(4) the enzyme required FAD for activity; FMN and riboflavin would not substitute for FAD. 4. The rate of anaerobic reduction of FAD by NAD(P)H was increased more than tenfold in the presence of 4-hydroxypyridine, suggesting that the mechanism of hydroxylation was similar to that of other aromatic hydroxylases which are of the mono-oxygenase type. 5. The partially purified enzyme was extremely specific for its heterocyclic substrate but would utilize either NADH or NADPH. 6. 4-Hydroxypyridine-3-hydroxylase was strongly inhibited by high substrate concentration (above 0.5mm) especially below pH7.5. 8. The inflexion at pH8.4 in a pK(m) versus pH plot, together with strong inhibition by p-chloromercuribenzoate, suggested a role for thiol groups in substrate binding.
D000645:Ammonium Sulfate; D002729:Chloromercuribenzoates; D002850:Chromatography, Gel; D002854:Chromatography, Paper; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011725:Pyridines / Q000378:metabolism*; D012231:Rhizobium / Q000378:metabolism*; D012256:Riboflavin / Q000378:metabolism; D013438:Sulfhydryl Compounds; D014461:Ultracentrifugation; D014465:Ultrasonics
D002729:Chloromercuribenzoates; D011725:Pyridines; D013438:Sulfhydryl Compounds; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005486:Flavin Mononucleotide; D006899:Mixed Function Oxygenases; D010100:Oxygen; D000645:Ammonium Sulfate; D012256:Riboflavin