Purification and characterization of the 3-hydroxybenzoate-6-hydroxylase from Klebsiella pneumoniae.
We isolated 3-hydroxybenzoate-6-hydroxylase (E.C.1.14.13.), an inducible enzyme that catalyzed the para-hydroxylation of 3-hydroxybenzoate (3-HBA) to 2,5-dihydroxybenzoate, from Klebsiella pneumoniae. Although the enzyme was found to be mainly induced by its substrate, a coordinated induction of 3-hydroxybenzoate hydroxylase and gentisate dioxygenase was also observed in the presence of the product of the reaction. The purified enzyme was a monomer with a molecular mass of 42,000. It contained FAD as a prosthetic group, utilized NADH or NADPH with similar efficiencies and its activity was inhibited by Cu2+, Fe2+ and Hg2+. Other properties, such as induction mechanism and kinetic parameters were also studied. Moreover, for the first time the amino acid composition of a 3-hydroxybenzoate-6-hydroxylase was determined.
Suárez
M
M
Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain.
Ferrer
E
E
Garrido-Pertierra
A
A
Martín
M
M
D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D002413:Cations, Divalent / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D049308:Dioxygenases; D004790:Enzyme Induction / Q000187:drug effects; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005841:Gentisates; D062385:Hydroxybenzoates / Q000378:metabolism; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010105:Oxygenases / Q000096:biosynthesis
D000596:Amino Acids; D001426:Bacterial Proteins; D002413:Cations, Divalent; D005841:Gentisates; D062385:Hydroxybenzoates; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D006899:Mixed Function Oxygenases; D010105:Oxygenases; D049308:Dioxygenases; C026247:gentisate 1,2-dioxygenase; C023315:3-hydroxybenzoate-6-hydroxylase; D004047:Diethyl Pyrocarbonate; C010925:2,5-dihydroxybenzoic acid