Genetics and enzymology of the B12 pathway

The chemical complexity of vitamin B12 suggests that its formation may involve a large number of enzymic steps. However, until recently, little was known of the number, mechanism and stereochemical course of the many enzymic interconversions that are essential to vitamin B12 biosynthesis. In response to this the French groups led by Francis Blanche and Joel Crouzet have carried out extensive investigations into the genetic and biochemical organization of this remarkable biosynthetic pathway. Through heterologous complementation studies with cobalamin-producing mutants they were able to clone and identify a total of 22 unique cob genes from four genomic regions (A-D) of the Pseudomonas denitrificans chromosome. This was the first report of a genetic analysis of cob genes at the molecular level and provided a suitable genetic model from which biosynthetic investigations could be initiated. The metabolic roles of most of the products of these genes have now been defined and in light of this progress current research concentrates on the development and use of a variety of techniques to investigate the chemistry involved in these individual enzymic steps. Here, my focus is on the recent efforts and successes of the French groups that have led to the elucidation of almost the entire enzymic sequence of events in vitamin B12 biosynthesis. From this perspective, recent developments at Cambridge (UK) regarding the utilization of reconstituted enzymic systems to manufacture substrates as probes for this biosynthetic pathway are illustrated.