Studies on vitamin B12 biosynthesis and on 2,3-Oxidosqualene cyclase

Abstract

Vitamin B 12, the antipemicious anemia factor, is known to be the most complex nonpolymeric natural product derived from uroporphyrinogen III. Its coenzyme is the tetrapyrrolic cofactor involved in over 20 enzymatic reactions. For the past half-century, the biosynthesis of vitamin B 12 has been investigated with great attention. Many chemists and biochemists have been interested in the ring contraction process to make a corrin. The efforts on the trial synthesis resulted in the Woodward-Hoffmann rules. This research gave the answer for the ring contraction mechanism in vitamin B 12 biosynthesis. The enzymes for oxidative ring contraction and insertion of the fourth methyl group at C-17 to produce precorrin 4, which is the first metal-free corrin intermediate in vitamin B 12 biosynthesis, were discovered. CobG from Pseudomonas denitrificans was cloned, overexpressed, purified and characterized. The ring contraction has been shown to involve the conversion of precorrin 3 to precorrin 3x ( a hydroxy lactone ) catalyzed by the Pseudomonas denitrificans enzyme CobG which is found to be dependent on molecular oxygen. The enzyme CobJ is a SAM-dependent C-17 methyl transferase, necessary for ring contraction. Also, CobM has been identified as the enzyme responsible for the C-methylation at C -l 1, leading to the isolation of precorrin 5, the last missing intermediate of the biosynthetic pathway between precorrin 3 and precorrin 6x. Using the first 8 enzymes of the pathway, the genetically engineered synthesis of precorrin 5 from ALA in a single flask has been accomplished successfully. Precorrin 6x was synthesized from the incubation using precorrin 4, CobM, CobF and SAM. Finally, CobK, precorrin 6x reductase, was cloned and overexpressed. Precorrin 6y was synthesized from the incubation containing precorrin 4, CobM, CobF, CobK, SAM and NADPH. The cyclization of oxidosqualene is one of the most remarkable steps in the biosynthesis of steroids and triterpenoids. 2,3-Oxidosqualene cyclase, a membrane bound enzyme, from Saccharomyces cerevisiae has been partially purified and stabilized. Candida albicans erg7 gene, which encodes oxidosqualene cyclase, was cloned, sequenced, and overexpressed. To overcom e inclusion body formation, solubilization and folding experiments were tried. Overexpression of erg7 gene in yeast expression and baculovirus expression systems was performed to get soluble overexpressed oxidosqualene cyclase.