S34: Unfavorable consequences of introducing a novel pathway into a pre-existing metabolic network

Promiscuous enzyme activities provide a starting place for the evolution of novel enzyme activities, both in nature and in the hands of protein engineers.  However, the potential of promiscuous enzymes extends beyond the evolution of single enzymes to serve new functions.  Microbes contain hundreds of enzymes, raising the possibility that promiscuous enzymes can be patched together to generate “serendipitous” pathways that are not part of normal metabolism.  We have elucidated the steps in a serendipitous pathway that allows E. coli to synthesize PLP when the normal biosynthetic pathway is blocked and either yeaB or thrB is overexpressed.  One of the unusual metabolites in this pathway, hydroxypyruvate, is toxic because it inhibits serine biosynthesis.  Furthermore, flux through the serendipitous pathway is inhibited by homoserine, which is the normal substrate for an enzyme that catalyzes a promiscuous reaction in the pathway.  These findings illustrate the unfavorable interactions that can take place between novel pathways and a pre-existing metabolic network.  These interactions are not surprising.  In the first instance, proteins do not evolve to discriminate against molecules that are typically not present in the cytoplasm.  In the second instance, the normal substrate of an enzyme will be a potent competitive inhibitor of any promiscuous reaction catalyzed at the same active site.  Evolutionary processes should be able to overcome both of these problems.