Bacterial metabolism of resorcinylic compounds: purification and properties of orcinol hydroxylase and resorcinol hydroxylase from Pseudomonas putida ORC.
The hydroxylase activities observed in extracts of Pseudomonas putida ORC after growth on orcinol and resorcinol as sole source of carbon have been purified to homogeneity. Both enzymes were shown to be flavoproteins and to contain approximately 1 mol of FAD for each polypeptide chain, S20,W values for each enzyme are 4.1 +/- 0.1 and are independent of the presence of their aromatic substrates. Molecular weight determinations under native (approximately 68000) and denaturing (approximately 70000) conditions indicated that they are monomeric. The visible absorption spectra identical but the circular dichroic spectra of the two proteins can be distinguished. Although each protein catalyzes the NAD(P)H and O2-dependent hydroxylation of both orcinol and resorcinol, the efficiency of the transformations of the substrates by the two enzymes is radically different; furthermore resorcinol hydroxylase is much more versatile in the aromatic compounds it can utilize as substrates and effectors. Other properties of the enzymes which clearly establish their own identity include their serological characteristics and amino acid composition; the latter property is particularly evident when the quantities of valine and alanine residues are compared. The synthesis of each enzyme is also under different regulatory constraints, being controlled by the substrate used for growth.
D000596:Amino Acids / Q000032:analysis; D002942:Circular Dichroism; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D006899:Mixed Function Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009243:NAD; D009249:NADP; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology*; D012118:Resorcinols / Q000378:metabolism; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D012118:Resorcinols; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D006899:Mixed Function Oxygenases