Figure 1. Models of enzyme sub-functionalization. When loss of a trpF gene occurs evolution of a proficient dual-substrate PriA enzyme takes place after positive selection. From that stage onwards, two possible scenarios can be envisaged. (A) The most accepted model of enzyme evolution implies gene duplication followed by narrowing of substrate specificity (subHisA and subTrpF), which is expected to occur after relaxation of the purifying selection. (B) Alternatively, differential gain of a trpF gene after HGT (marked with a lightning bolt), which does not imply the genetic and functional redundancy that is generated after gene duplication, could also take place. Thus, the sub-functionalization of PriA is constrained since purifying selection for the original function, i.e., HisA, is still in place. Moreover, the need for integrating the HGT-acquired whole-pathway trp operon into the receiving metabolic network, by means of altering substrate specificity, implies positive selection.