Purification and characterization of 4-hydroxybenzoate 3-hydroxylase from a Klebsiella pneumoniae mutant strain.
Unlike the parent wild-type strain, the Klebsiella pneumoniae mutant strain MAO4 has a 4-HBA+ phenotype. The capacity of this mutant to take up and metabolize 4-hydroxybenzoate (4-HBA) relies on the expression of a permease and an NADPH-linked monooxygenase (4-HBA-3-hydroxylase). Both enzymes are normally expressed at basal levels, and only the presence of 4-HBA in the media enhances their activities. Strikingly, when the Acinetobacter calcoaceticus pobA gene encoding 4-hydroxybenzoate-3-hydroxylase was expressed in hydroxybenzoate K. pneumoniae wild-type, the bacteria were unable to grow on 4-HBA, suggesting that the main difference between the wild-type and the mutant strain is the capability of the latter to take up 4-HBA. 4-HBA-3-hydroxylase was purified to homogeneity by affinity, gel-filtration, and anion-exchange chromatography. The native enzyme, which appeared to be a dimer of identical subunits, had an apparent molecular mass of 80 kDa and a pI of 4.6. Steady-state kinetics were analyzed; the initial velocity patterns were consistent with a concerted substitution mechanism. The purified enzyme had 362 amino acid residues, and a tyrosine seemed to be involved in substrate activation.
Suárez
M
M
Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain.
Martín
M
M
Ferrer
E
E
Garrido-Pertierra
A
A
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000150:Acinetobacter / Q000201:enzymology / Q000235:genetics; D000596:Amino Acids / Q000032:analysis; D004047:Diethyl Pyrocarbonate; D004790:Enzyme Induction; D005798:Genes, Bacterial / Q000235:genetics; D007526:Isoelectric Point; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D008970:Molecular Weight; D009154:Mutation; D010226:Parabens / Q000378:metabolism; D014169:Transformation, Bacterial
D000596:Amino Acids; D010226:Parabens; D015109:4-Hydroxybenzoate-3-Monooxygenase; C038193:4-hydroxybenzoic acid; D004047:Diethyl Pyrocarbonate