PURIFICATION AND CHARACTERIZATION OF ACINETOBACTER-CALCOACETICUS 4-HYDROXYBENZOATE 3-HYDROXYLASE AFTER ITS OVEREXPRESSION IN ESCHERICHIA-COLI

被引：3

作者：

FERNANDEZ, J

DIMARCO, AA

ORNSTON, LN

HARAYAMA, S

机构：

[1] UNIV GENEVA,MED CTR,DEPT MED BIOCHEM,CH-1211 GENEVA,SWITZERLAND

[2] YALE UNIV,DEPT BIOL,NEW HAVEN,CT 06511

来源：

JOURNAL OF BIOCHEMISTRY | 1995年 / 117卷 / 06期

关键词：

ACINETOBACTER CALCOACETICUS; DIHYDROFOLATE REDUCTASE; 4-HYDROXYBENZOATE; 3-HYDROXYLASE; OVEREXPRESSION; POBA;

D O I：

10.1093/oxfordjournals.jbchem.a124853

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

4-Hydroxybenzoate 3-hydroxylase [EC 1.14.13.2] from Acinetobacter calcoaceticus was purified to homogeneity following the 40-fold overexpression of this gene (pobA) in Escherichia coli. Overexpression was accomplished by placing the folA gene (encoding trimethoprim-resistant dihydrofolate reductase) directly downstream of the pobA gene, and demanding growth of recombinants on elevated concentration of trimethoprim. Presumably, the surviving variants have undergone a genetic alteration which allowed the overexpression of both folA and pobA. 4-Hydroxybenzoate 3-hydroxylase was purified in two chromatographic steps, characterized biochemically, and its properties were compared to those of its homolog from Pseudomonas fluorescens. The two enzymes differ in their reponse to Cl- ion inhibition. A single amino acid change in the putative NADPH-binding site is proposed to account for this difference. The inhibitory and catalytic properties of substrate analogs were also examined.

引用

页码：1261 / 1266

页数：6

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