Studies of the ribosome-associated vitamin B12S adenosylating enzyme of Lactobacillus leichmannii.

The ribosomes of Lactobacillus leichmannii (ATCC 7830) are the loci of an enzyme system that converts vitamin B₁₂ (cyanocobalamin, CN-Cbl) to adenosylcobalamin (AdoCbl) in two steps, reduction of B_12a (Co III) to B_12s (Co I) by B_12a reductase and the adenosylation of B_12s to AdoCbl by an adenosylating enzyme. The vitamin B₁₂ reductase, as in other organisms, is unstable. Adenosylating enzyme, however, is readily demonstrable. Reported experiments deal primarily with that enzyme. Evidence of the association between ribosomes and adenosylating enzyme was found in sucrose density gradient analyses. Intact, washed ribosomes yielded an enzyme activity profile that coincided with the ultraviolet maximum of 70S reference ribosomes. When intact ribosomes were exposed to 2.5 m CsCl so that 70% of ribosomal protein was recoverable in the 144,000g supernatant fraction and >90% of RNA was in the pellet, adenosylating enzyme was found in the supernatant fraction. “Stripped” ribosomes had low levels of enzyme activity and could reassociate with free enzyme protein. Stripped ribosomes remained competent in protein synthesis. Hence, adenosylating enzyme is not an integral ribosome component. Partially purified ribosome-associated B_12s adenosylating enzyme has requirements for vitamin B_12s, ATP, and Mn²⁺, though Mn²⁺ could be partially replaced by Mg²⁺. Isotopic studies showed that ATP is the source of the adenosyl moiety of AdoCbl and that inorganic tripolyphosphate is a reaction product. Substantial adenosylating activity is associated with ribosomes only in the vitamin B₁₂-requiring lactobacilli, L. delbrueckii (ATCC 9649) and L. leichmannii. Surprisingly, L. casei (ATCC 9595) ribosomes displayed a measurable, if low, level of activity. L. acidophilus (ATCC 11506) ribosomes had no detectable activity. The bulk of the activity in Clostridium tetanomorphum (ATCC 3606) and Propionibacterium shermannii (ATCC 9614) is in the 144,000g supernatant fraction. Ribosomes from animal cells (liver, reticulocytes, and Ehrlich ascites tumor) were without detectable activity.