PMID:8399388
| Citation |
Bugg, TD (1993) Overproduction, purification and properties of 2,3-dihydroxyphenylpropionate 1,2-dioxygenase from Escherichia coli. Biochim. Biophys. Acta 1202:258-64 |
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| Abstract |
The mhpB gene encoding 2,3-dihydroxyphenylpropionate 1,2-dioxygenase in Escherichia coli was subcloned from Clarke-Carbon plasmid pLC20-30 by complementation with an mhpB- strain LW366. Dioxygenase MhpB was purified using a five-step procedure from an overexpressing construct containing the mhpB gene, giving enzyme of > 95% homogeneity. The purified enzyme appeared as a 36-kDa subunit by SDS-PAGE, and had a native molecular mass of 134 kDa as determined by gel filtration. The apoenzyme obtained after chromatography could be re-activated by addition of Fe(II) and ascorbate to give the holoenzyme with a specific activity of 48 U/mg, which could be readily inactivated by oxidation or complexation of the Fe(II) cofactor, or simply by dilution. The substrate specificity of MhpB was examined, and as well as 2,3-dihydroxyphenylpropionate the enzyme was found to catalyse meta-ring cleavage of 3-methylcatechol and catechol, with reduced catalytic efficiency. The N-terminal sequence obtained for the purified enzyme showed significant sequence similarity with catechol 2,3-dioxygenase from Alcaligenes eutrophus, but none with catechol 2,3-dioxygenases from Pseudomonas. |
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| Keywords |
Amino Acid Sequence; Ascorbic Acid; Cloning, Molecular; Dioxygenases; Escherichia coli/enzymology; Escherichia coli/genetics; Molecular Sequence Data; Oxygenases/biosynthesis; Oxygenases/chemistry; Oxygenases/isolation & purification; Recombinant Proteins/biosynthesis; Substrate Specificity |
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