In sum although it is reasonable to assume that the
In sum, although it is reasonable to assume that the activities of an enzyme on alternative (metabolically available) substrates may often depend on neutral drift, the point is difficult to prove because various types of selective pressures can be at play, and these will be different in different organismal lineages 57, 58. For many enzymes, the boundaries between an evolutionarily selected degree of substrate promiscuity and an evolutionarily irrelevant degree of sloppiness may be difficult to establish (See Outstanding Questions).
Substrate Promiscuity Contributes to Underground Metabolism As seen in the sections above, enzymes have an unavoidable tendency to act on alternative, available substrates. Even when (perhaps not very often) this tendency is restrained to near the minimum level allowed by chemistry, and even in the presence of ‘repair’ systems, these enzymes will generate alternative products, many of which will be nonstandard metabolites. Substrate promiscuity must be hence considered as a major contribution to the complexity of the metabolome – together with catalytic promiscuity and other factors such as the non-enzymatic transformation of metabolites and the introduction of xenobiotics 44, 45. The contribution of substrate promiscuity is in part predictable , and understanding it may be necessary for thorough mapping the metabolome, one of the current challenges of biomolecular sciences . The compounds generated by side activities of metabolic enzymes sometimes contribute to a subterranean ensemble of reactions, proceeding in general at extremely low fluxes, that are collectively termed ‘underground metabolism’ 61, 62. It is believed that this messy network of reactions, while somehow inevitable, may represent a basis for the further evolution and diversification of metabolism 54, 63. That messiness may be central to the evolution of new beneficial functions is well exemplified by many pathways in secondary (specialized) metabolism. In gaba receptors to primary metabolism, specialized metabolism contributes to fitness by producing molecules that interfere with the biology of other organisms (e.g., environmental competitors). Many pathways in specialized metabolism tend to include multiple enzymes with non-strict substrate specificity and to produce an entire suite of chemically similar, but distinct, compounds 64, 65, 66. The most direct advantage, in this case, is that the ability to generate a multiplicity of secondary metabolites offers more flexibility in responding to different, and evolving, organisms in the environment. In primary metabolism, the ‘messy’ underground reactions could also, in some cases, facilitate evolutionary adaptation to new environments or provide bypass to genetic lesions 61, 62. For example, Kim et al. found that deletion of gene PdxB, that is required in E. coli for biosynthesis of pyridoxal phosphate, could be bypassed by at least three ‘underground’ routes (one of which was elucidated in detail, and was found to involve the promiscuous activity of homoserine kinase on an alternative substrate) . A fourth route was reported by another group, using promiscuity prediction software . Earlier, Patrick and coworkers had found that 41 of 104 E. coli knockout strains, that were unable to grow on minimal medium, could be rescued by overexpression of at least one gene different from the deleted gene. In several cases, the multicopy suppressor was not homologous to the deleted gene, and its effect was attributed to a promiscuous activity of the encoded enzyme or to a metabolic pathway bypass . Underground reactions may also become – at some point and in some organisms – incorporated into mainstream primary metabolism. A possible example is the promiscuous activity of phosphoglycerate dehydrogenase with α-ketoglutarate, yielding D-2-hydroxyglutarate. In humans, D-2-hydroxyglutarate is an oncometabolite  and requires a repair enzyme to be removed . In bacteria such as E. coli, however, the promiscuous activity of phosphoglycerate dehydrogenase is enhanced and is tightly coupled to the repair activity to thermodynamically drive L-serine biosynthesis  (Figure 3).