title: Catechol 1,2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259
Creators: name: Strachan, Philip
Publisher: name: University of Glasgow
Year: 1997
Subjects: subject: Microbiology.

description: 1. The Gram-positive bacterium Rhodococcus rhodochrous NCIMB13259 is able to grow on styrene, which it assimilates via a meta-cleavage pathway. This bacterium also contains a catechol 1, 2-dioxygenase that carries out intradiol-cleavage of aromatic substrates. 2. A rapid and reliable method for the purification of catechol 1, 2-dioxygenase was developed which involved chromatography on DEAE-Sephacel and phenyl- Sepharose. This procedure yielded at least 10 mg of protein from 15-20 g (wet weight) of cells. SDS-PAGE analysis of the final preparation showed that catechol 1, 2-dioxygenase was virtually homogeneous. The enzyme was stable and could be stored at -20°C with little loss in activity. 3. The enzyme was crystallised using 15% (w/v) polyethylene glycol 4 000, 0.33 M CaCl2, 0.25 mM Tris/HCl, pH 7.5, using sitting drop vapour diffusion. A microseeding technique was then used to obtain single, diffraction quality rectangular plates, which reached a size of 0.5 mm down the longest axis. A preliminary X-ray crystallographic characterisation showed that the crystals are in space group C2 with unit cell dimensions (A) a=111.9, b=78.1, c=134.6, ?=100°, with four subunits in the asymmetric unit. 4. A subunit Mr value of 39 800 was obtained with SDS-PAGE, which is approximately 20% more than the value of 31 558 which was obtained with electrospray mass spectroscopy. Similar overestimates by SDS-PAGE have also been obtained for other catechol 1, 2-dioxygenases. The native Mr value of 120 0(X) obtained by gel-peimeation chromatography indicated that catechol 1, 2-dioxygenase is a tetramer, however, catechol 1,2-dioxygenase which had been chemically cross- linked, showed only two bands upon analysis with PAGE, indicating that the enzyme is a dimer, with a calculated native Mr value of 63 000. Considerable evidence accrued by other workers with several non-haem intradiol-cleavage enzymes, indicates that the overestimate could be due to the catechol 1,2- dioxygenase being more elliptical than the protein standards, causing it to move anomolously through gels. 5. The absorbance spectrum of catechol 1,2-dioxygenase had maxima at 220 and 280 nm. At higher concentrations of the enzyme another maximum at 426 nm was also evident. This is good evidence that the Rhodococcus enzyme is iron dependent, since in several non-haem iron intradiol-cleavage enzymes a broad absorption band at about 440 nm is due to an Fe3+ cofactor. 6. Iron quantitation using colorimetry and atomic absolution spectroscopy found significantly less than the expected one mole of iron per subunit (0.60-0.85 atoms per subunit). 7. The activity and stability of catechol 1,2-dioxygenase as a function of pH were investigated. Enzyme activity increased approximately four-fold from pH 5 and reached an optimum value at approximately pH 9. Incubation of the enzyme for 30 min in solutions which ranged from pH 5 to 9, resulted in no significant loss of activity. At pH 11.2 however, the enzyme was rapidly inactivated. (Abstract shortened by ProQuest.).