PURIFICATION AND CHARACTERIZATION OF CATECHOL 1,2-DIOXYGENASE FROM RHODOCOCCUS-RHODOCHROUS NCIMB-13259 AND CLONING AND SEQUENCING OF ITS CATA GENE

A method was developed for the purification of catechol 1,2-dioxygenas e from Rhodococcus rhodochrous NCIMB 13259 that had been grown in the presence of benzyl alcohol. The enzyme has very similar apparent K-m ( 1-2 mu M) and V-max (13-19 units/mg of protein) values for the intradi ol cleavage of catechol, 3-methyl-catechol and 4-methylcatechol and it is optimally active at pH 9. Cross-linking studies indicate that the enzyme is a homodimer. It contains 0.6 atoms of Fe per subunit. The en zyme was crystallized with 15% (w/v) poly(ethylene glycol) 4000/0.33 M CaCl2/25 mM Tris (pH 7.5) by using a microseeding technique. Prelimin ary X-ray characterization showed that the crystals are in space group C2 with unit-cell dimensions a = 111.9 Angstrom, b = 78.1 Angstrom, c = 134.6 Angstrom, beta = 100 degrees. An oligonucleotide probe, made by hemi-nested PCR, was used to clone the gene encoding catechol 1,2-d ioxygenase (catA). The deduced 282-residue sequence corresponds to a p rotein of molecular mass 31539 Da, close to the molecular mass of 3155 8 Da obtained by electrospray MS of the purified enzyme. catA was subc loned into the expression vector pTB361, allowing the production of ca techol 1,2-dioxygenase to approx. 40 % of the total cellular protein. The deduced amino acid sequence of the enzyme has 56 % and 75 % identi ty with the catechol 1,2-dioxygenases of Arthrobacter mA3 and Rhodococ cus erythropolis AN-13 respectively, but less than 35 % identity with intradiol catechol and chlorocatechol dioxygenases of Gram-negative ba cteria.