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首页> 外文期刊>Archives of microbiology >PURIFICATION AND CHARACTERIZATION OF 4-HYDROXYBENZOATE 3-HYDROXYLASE FROM A KLEBSIELLA PNEUMONIAE MUTANT STRAIN
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PURIFICATION AND CHARACTERIZATION OF 4-HYDROXYBENZOATE 3-HYDROXYLASE FROM A KLEBSIELLA PNEUMONIAE MUTANT STRAIN

机译：肺炎克雷伯氏菌突变株中4-羟基苯甲酸酯3-羟化酶的纯化和鉴定

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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 hydroxy benzoate K. pneumoniae wild-type, the bacteria were un able 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 pl 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. [References: 29]
机译：与亲本野生型菌株不同，肺炎克雷伯氏菌突变菌株MAO4具有4-HBA（+）表型。该突变体吸收和代谢4-羟基苯甲酸酯（4-HBA）的能力取决于渗透酶和NADPH连接的单加氧酶（4-HBA-3-羟化酶）的表达。两种酶通常以基础水平表达，并且只有4-HBA存在于培养基中才能增强其活性。令人惊讶的是，当编码4-羟基苯甲酸酯-3-羟化酶的不动杆菌钙pobA基因在羟基苯甲酸酯肺炎克雷伯菌野生型中表达时，细菌无法在4-HBA上生长，这表明野生型之间的主要区别突变株是后者吸收4-HBA的能力。通过亲和力，凝胶过滤和阴离子交换层析将4-HBA-3-羟化酶纯化至均质。看起来是相同亚基的二聚体的天然酶具有80kDa的表观分子量和4.6的pI。分析了稳态动力学;初始速度模式与协调替代机制一致。纯化的酶具有362个氨基酸残基，酪氨酸似乎与底物活化有关。 [参考：29]

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