An activity-resistance tradeoff constrains enzyme evolution

The presence of self-resistance genes in antibiotic-producing organisms poses a paradox: how can resistance evolve before the antibiotic exists, and how can an antibiotic producer arise without first evolving resistance? Here we examine the evolutionary origins of self-resistance to mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase (IMPDH). The MPA biosynthetic gene cluster (BGC) includes a resistant IMPDH-B. Homologs of IMPDH-B occur not only in MPA producers but also in many non-producing fungi, where remnants of the MPA BGC remain detectable. The phylogeny of IMPDH-B is incongruent with the fungal species tree, consistent with multiple horizontal gene transfer events between Aspergillus and Sordariomycetes. We characterized eleven extant IMPDH-Bs, five from MPA producers and six from nonproducers, along with seven resurrected ancestral enzymes (Anc1-Anc7). MPA resistance appeared between Anc2 and Anc3 and coincided with a loss of catalytic efficiency. Across both ancestral and extant enzymes, MPA resistance correlated strongly with reduced activity, revealing a robust activity-resistance trade-off that has persisted for millions of years. Unexpectedly, both the IMPDH-Bs and ancestral enzymes Anc3-Anc7 were also resistant to ribavirin-5'-monophosphate (RVP), an IMP-competitive inhibitor. Because MPA and RVP bind to similar enzyme conformations, the activity-resistance trade-off likely reflects a design constraint imposed by the need to maintain resistance to multiple inhibitors. Intriguingly, although Anc1 and Anc2 are equally sensitive to MPA, Anc2 shows reduced susceptibility to RVP. This pattern suggests that pre-existing resistance to another IMPDH inhibitor may have created a permissive background for the later evolution of MPA biosynthesis. SignificanceAntibiotic producers must be resistant to the toxins that they produce, but how such self-resistance develops is a mystery. The mycophenolic acid (MPA) biosynthetic gene cluster (BGC) encodes a resistant variant of the MPA target IMPDH (IMPDH-B). Many fungi retain IMPDH-B although they have lost the ability to produce MPA. The IMPDH-B and species phylogenies are incongruent, suggesting evolution of the BGC was complicated. MPA resistance correlates with low catalytic efficiency in modern and ancestral IMPDHs, revealing a robust design constraint tradeoff. Surprisingly, IMPDH-Bs are also resistant to an IMP-competitive inhibitor (RVP). RVP resistance appears to have emerged before MPA resistance. Perhaps resistance to RVP created a background that permitted the genesis of a new toxin.