OVERPRODUCTION, PURIFICATION AND PROPERTIES OF 2,3-DIHYDROXYPHENYLPROPIONATE 1,2-DIOXYGENASE FROM ESCHERICHIA-COLI

The mhpB gene encoding 2,3-dihydroxyphenylpropionate 1,2-dioxygenase i n Escherichia coli was subcloned from Clarke-Carbon plasmid pLC20-30 b y complementation with an mhpB- strain LW366. Dioxygenase MhpB was pur ified using a five-step procedure from an overexpressing construct con taining the mhpB gene, giving enzyme of > 95% homogeneity. The purifie d enzyme appeared as a 36-kDa subunit by SDS-PAGE, and had a native mo lecular 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 complexati on of the Fe(II) cofactor, or simply by dilution. The substrate specif icity of MhpB was examined, and as well as 2,3-dihydroxyphenylpropiona te the enzyme was found to catalyse meta-ring cleavage of 3-methylcate chol and catechol, with reduced catalytic efficiency. The N-terminal s equence obtained for the purified enzyme showed significant sequence s imilarity with catechol 2,3-dioxygenase from Alcaligenes eutrophus, bu t none with catechol 2,3-dioxygenases from Pseudomonas.