Catalytic versus inhibitory promiscuity in cytochrome P450s: implications for evolution of new function.

Promiscuity at the molecular level plays a critical role in several cellular processes including signal transduction, immunity, and detoxification (1–9). More broadly, the evolution of new enzymatic activities from a pool of existing protein scaffolds, whether in nature or in vitro directed evolution, may proceed through ‘promiscuous’ intermediates that catalyze multiple reactions (10–19). A common description of this possible evolutionary process and relevant factors is schematized in Figure 1, wherein a specific enzyme undergoes mutation to yield a promiscuous variant that catalyzes a new reaction, without forfeiting its original activity (11, 20). At early stages along the evolutionary trajectory the mutation is neutral with respect to the original function. If this second enzymatic activity provides any survival advantage, then it is retained and, after gene duplication, one copy of the gene encoding the promiscuous enzyme can undergo further refinement to optimize the new function, without loss of the original function. It may be useful to distinguish between ‘promiscuous’ enzymes with catalytic activities unrelated to their presumed biological function, and ‘multifunctional’ enzymes with a clear functional advantage in accepting multiple substrates, as suggested (20). Here we use the term ‘promiscuity’ for both cases, because we aim to understand the physical and chemical properties of enzymes that process multiple substrates, regardless of the functional purpose. Recent studies have suggested that promiscuous intermediates easily evolve into efficient and specific enzymes (11, 20). While this model provides an extremely useful framework to conceptualize evolutionary structure-function relationships, ‘promiscuity’ is not a well-understood property, and it is difficult to quantify, although progress has been made (17, 19). Figure 1 A. Promiscuous Intermediate Model for Evolution of New Enzymatic Function. Enzymes with high catalytic specificity for one reaction (original catalytic activity) move along the indicated trajectory. At early stages they become promiscuous, gaining new ... For example, natural evolution of new function does not happen in isolation. Rather, each evolutionary intermediate must function in the context of the existing niche that includes other enzymes and a ‘background’ of potential ligands. Within a niche, a loss due to mutation in any useful trait is referred to as a ‘negative trade-off’(20). The evolutionary trajectory of an enzyme is unlikely to be a two dimensional process as depicted in the model in Figure 1a, but it will trace a multidimensional surface that includes the relationships between promiscuity and other properties that comprise the overall fitness of the enzyme. That is, there will be a third collective coordinate for negative trade-offs, as in Figure 1b. In this case, ‘promiscuity’ per se is not an obvious advantage in the evolutionary process unless it is achieved without significant negative trade-offs (20). Whereas a highly promiscuous enzyme would provide a very efficient template from which to evolve many new functions from a single protein sequence, such an intermediate would unlikely be neutral with respect to all of the original properties. If the magnitude of any negative trade-off is coupled exactly to the magnitude of promiscuity, then the mutational neutrality will not be maintained in all dimensions of fitness space and the path towards new function will have a different shape than if the putative negative trade-off is not correlated with the promiscuity in any dimension. Ultimately, the relationship between any potential negative trade-off and catalytic promiscuity will determine whether evolution will follow that path. These concepts underscore the need to define the relationship between catalytic promiscuity and other protein properties. Speculatively, for example, a new promiscuous enzyme would have increased susceptibility to inhibition by ligands that do not inhibit the ‘original’ enzyme and this would be a negative trade-off. This is based on the observation that structurally diverse drugs can bind to and inhibit a single enzyme, without yielding products (consider antibiotics, statins, etc.). In contrast catalysis requires proper alignment of multiple reactive functional groups. Each incremental step towards promiscuity, i.e. towards the diagonal of Figure 1a, will only be advantageous if the new catalytic activity is not accompanied by an equal or greater increase in susceptibility to ‘new’ inhibitory interactions that, at ambient ligand concentrations, limit the original enzyme activity. For the example here, inhibitory promiscuity is a potential negative trade-off that adds a new dimension to yield the scheme in Figure 1b, and can be substituted by any parameter hypothesized to be a negative trade-off. We emphasize that the current studies are not intended to establish specific evolutionary mechanisms, which may best be accomplished by successive rounds of random mutagenesis and relevant selection protocols. Rather we aim to understand more generally the relationships between catalytic promiscuity and other properties of enzymes that could limit or define evolutionary pathways. An understanding of these relationships is necessary to understand the role of catalytic promiscuity in evolution and to advance the incomplete model of Figure 1. The Cytochrome P450s provide an excellent test case to further understand these relationships because, as a family with highly conserved scaffold, they span a very wide range of catalytic promiscuity. Human hepatic CYPs dominate drug metabolism and have very broad substrate selectivities (19, 21). Here we use these multifunctional CYPs as a model for physico-chemical traits of enzymes near the limits of catalytic promiscuity. Other CYPs, including bacterial and human isoforms used here, contribute to specific biosynthetic pathways and are very substrate specific (22, 23). Collectively, the CYP superfamily provides insight into the relationship between catalytic and inhibitory promiscuity. The results indicate a surprising level of inhibitory promiscuity even for substrate-specific CYPs, and they further suggest that because inhibitory promiscuity is already present in highly specific enzymes, there is little cost associated with evolutionary steps toward catalytic promiscuity.