| Aspartate transcarbamoylase (ATCase) from different bacteria such as Mycobacterium, Burkholderia cepacia pyrB clone, Burkholderia cepacia wild type, Synechocystis and some other pathogen bacteria were characterized and studied its interaction with other pyrimidine enzymes. ATCase and DHOase of these bacteria were assayed and their enzyme was purified using conventional methods and different column such as sphacryl, DEAD.MonoQ, HIC and HPLC. In Mycobacterium the two activities (ATCase and DHOase) were found to be copurified and ATCase was active not as a dodecamer, the way which is in the Pseudomonas or Sterptomces. Other microorganism that has the same activity is Deinococcus. The holoenzyme of M. smegmatis was 480 kDa and 390 kDa and M. phlei holoenzyme was 480, 450, 410 and 380 kDa. Both Mycobacterium in SDS-PAGE gel reveal two subunit, 35 and 45 kDa. Other organisms that have pyrC active are Streptomysis, Thermus and Burkholderia cepacia. Mycobacterium ATCase showed typical Michaelis-Menten kinetics for velocity versus substrate plots for aspartate and their ATCase was inhibited by ATP, CTP, and UTP. These observations place the Mycobacterium ATCase in class A groups.; B. cepacia is the other organisms, which was characterized its pyrimidine enzyme. After Dr farinha prepare a clone of B. cepacia pyrB, into E. coli TB2, its enzyme has been characterized by this study.; The result shows that ATCase in this organism can be active as a trimer, the same which was on Bacillus groups. This pyr B polypeptide has been purified and sent to the Davis UCL for Edman degradation and the amino acid sequence match perfectly with the DNA sequence, which Farinha prepared. For comparison the B. cepacia wild type also has been purified. The results in nondenaturing gel showed that B. cepacia ATCase is composed of two subunits, the large polypeptide (550 kDa) and small polypeptide (165 kDa). The holoenzyme is composed of two subunit, 52 and 38 kDa.; The ATCase size from photosynthetic bacteria such as Synechocystis and Synechococcus has been found that is Pseudomonas size (480kDa). ATCase size from one alga, Agmonellum has been found that is the same size of B. cepacia (550 kDa). |
