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-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 that had been grown in the presence of benzyl alcohol. The enzyme has very similar apparent Km (1-2 microM) and Vmax (13-19 units/mg of protein) values for the intradiol cleavage of catechol, 3-methylcatechol and 4-methylcatechol and it is optimally active at pH9. Cross-linking studies indicate that the enzyme is a homodimer. It contains 0.6 atoms of Fe per subunit. The enzyme was crystallized with 15% (w/v) poly(ethylene glycol) 4000/0.33 M CaCl2/25 mM Tris (pH7.5) by using a microseeding technique. Preliminary X-ray characterization showed that the crystals are in space group C2 with unit-cell dimensions a=111.9 A, b=78.1 A, c=134.6 A, beta=100 degrees. An oligonucleotide probe, made by hemi-nested PCR, was used to clone the gene encoding catechol 1,2-dioxygenase (catA). The deduced 282-residue sequence corresponds to a protein of molecular mass 31539 Da, close to the molecular mass of 31558 Da obtained by electrospray MS of the purified enzyme. catA was subcloned into the expression vector pTB361, allowing the production of catechol 1,2-dioxygenase to approx. 40% of the total cellular protein. The deduced amino acid sequence of the enzyme has 56% and 75% identity with the catechol 1, 2-dioxygenases of Arthrobacter mA3 and Rhodococcus erythropolis AN-13 respectively, but less than 35% identity with intradiol catechol and chlorocatechol dioxygenases of Gram-negative bacteria.

Correspondence (Infectiousness of neural cases)

While from the nature of things it is impossible to prove that patients whose lesions are of the bacteriologically negative, resistant type are under no circumstances able to trasmit leprosy, available evidence is against their being a source of danger to children, who are usually highly susceptible. With regard to the juvenile type, the frequency with which such cases suddenly develop widespread, bacteriologically positive lesions makes clear the necessity of keeping children suffering from this type of the disease under close observation, remembering the danger of their suddenly becoming actively infectious cases. THere is a tendency for advanced cutaneous-type cases to develop nerve lesions in the later stages of the disease. Patients seen at this stage are often classified as neural (properly, " secondary neural"). Routine bacteriological examination may at first fail to show Mycobacterium leprae, but repeated examination will often show clumps of acid-fast bacilli in the skin, nose or gums. Patients who have at one time suffered from widespread sutaneous leprosy with bacteriologically positive lesions should, even after the disease has become quiescent and arrested, be kept from close contact with healthy people and especially with children. Such contact should be permitted only when the disease has remained arrested for several years, when the patients have shown no signs of the disease on being subjected to the iodice test, and when all parts of the skin, nasal mucosa, gums, etc., have been carefully examined for bacteria and found negative. Reference is made to the use of the leprolin test in determining resistance to invasion by Myco. leprae and in judging the degree of danger of transmission of disease.