243. 4-Methoxycinnamylidenepyruvic acid

Microbial Biosynthesis of B12-Like Compounds

“…The GDP-cobinamide was labeled with 3 2 P. The very small activity, twice the experimental counting error, corresponded to a specific activity of 0.3 mp,moles product per hr per mg protein at 37°C. Since it is now known that vitamin B1 2 5'-phosphate appears to be an obligatory precursor of the vi tamin in P. shermanii (94), and that a reaction presumably takes place with a-ribazole 5'-phosphate rather than with the nucleoside, these low activities are perhaps not to surprising. However, Renz (230 ), on the basis of color imetric assays of products, reports activities in crude dialyzed P. shermanii extracts for reaction between GDP-cobinamide and a-ribazole 5'-phosphate which appear to be only little higher than those found by Ronzio & Barker with a-ribazole.…”

“…There are at least three enzymatic steps between free base and final cobamide : formation of N-o:-glycosidic 5' -nucleotide, reaction with GDP-cobinamide, dephosphory lation. The only example of base incorporation beside DBI for which there is some evidence for each of these three steps is that of 5,6-dichlorobenzimi dazole (94). If this pathway is in d eed the only one operative in de novo incorporation of bases then none of these three enzymes can reject the base.…”

“…Enzymatic details are discussed further be low. The role of the traces of uncharacterized incomplete acidic corrinoids observed after addition of benzimidazoles to various microorganisms (94) is obscure.…”

“…3) . A new corrinoid, present at a low steady-state concentration, is formed as an obligatory intermediate and was identified chemically as vitamin B12 5' -phosphate (94) . The isolation was helped by the fortuitous observation that heat treatment of the bacteria in creases the amounts of the substance.…”

“…The isolation was helped by the fortuitous observation that heat treatment of the bacteria in creases the amounts of the substance. Upon acid hydrolysis the corrinoid yields the 3',5' -diphosphate of DB I-nucleoside (94) . The identification of the corrinoid as vitamin B12 5'-phosphate was confirmed by X-ray analysis of the crystals obtained after purification from extracts of heated P. sh ermanii (70).…”