Amidation of,and (R)-1-amino-2-propanol attachment to,the corrin ring during vitamin B-12 biosynthesis by Clostridium tetanomorphum extracts

Two intermediate stages in cobalamin biosynthesis, amidation of carboxylic acid groups in the corrin ring and (R)-1-amino-2-propanol attachment at propionic acid position ?, have been studied using cell-free extracts from the obligate anaerobe Clostridium tetanomorphum. The preparation of an incomplete corrinoid, probably cobinic acid-a,c,d,e,g-pentaamide, as an in vitro amidation substrate was accomplished via mild acid hydrolysis of cobinamide. Weak, but reproducible activities for both amidation and (R)-1-amino-2-propanol attachment were found in crude, nucleic acid-free and DE-52 column-purified protein fractions. The amidation reaction was glutamine-dependent in crude fractions, but became ammonium ion-dependent in more purified fractions. Significant problems encountered were (a) the weak and unstable character of both enzyme activities, and (b) the irreversible changes in the visible spectra of the incomplete corrinoids employed as substrates caused by use of thiol-reducing agents in the buffers and assays.