Cloning the CobN Subunit of Cobalt Chelatase Gene Fragment from Pseudomonas aeruginosa Local Strain

• Main Author: OKSRI DONA (NIM : 10513058), LUSI
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/28665
• Institution: Institut Teknologi Bandung

<p align="justify">Vitamin B12 (cobalamin) is one of the most common cofactors synthesized by bacteria. This vitamin has an important function in maintaining the normal activity the brain, nervous system, and the formation of red blood cells for human. Biosynthesis of vitamin B12 includes the insertion of cobalt in the tetracyclic pyrole group consisting of a corrin ring. The insertion of this cobalt occurs enzymatically, catalyzed by cobalt chelatase. Cobalt chelatase is an ATP-dependent enzyme consisting of three subunits: CobS, CobT, and CobN. The CobN subunit has a binding site for hydrogenobirinic acid-a,c-diamides and cobalt ions, that has a function to enhance the activity of cobalt chelatase. The hydrogenobyrocinic acid-a,c-diamide is a natural substrate for cobalt chelatase, wich is composed that of tetracyclic pyrole group with four nitrogen atoms in the center of the corin ring and two ligands form a complex molecule with cobalt atom. Pseudomonas aeruginosa is one of the bacteria that can produce cobalt chelatase. In this study, a clone fragment of cobalt chelatase of CobN subunit from Pseudomonas aeruginosa local strain was obtained by PCR approach using R2linbB primer (5'-GGCGATTGCGCCCAGCATAA-3 ') as a forward and reverse primers. The fragment of the isolated CobN fragment consists of 730 base pairs, obtained from as a recombinant clone in pGEM_fCobN in E. coli TOP host. This fragment codes for 242 amino acids which have 99% homology with partial cobalt chelatase from Pseudomonas aeruginosa, with one difference of amino acid L511Q. Compared with the cobalt chelatase CobN subunit from Pseudomonas aeruginosa strain PA103, the fCobN gene codes amino acids residues from 471 to 716 compare to 1248 total amino acids in cobalt chelatase.<p align="justify">