Harnessing Promiscuity Patterns to Map Evolution in the Alkaline Phosphatase Superfamily

Abstract

In the past 30 yeas scientists have demonstrated that many enzymes are capable of promiscuous catalytic activity, in which one enzyme catalyses the turnover of chemically distinct substrates. It has been suggested that such promiscuity can play an important role in the evolution of enzyme function. This phenomenon is particularly pronounced in the alkaline phosphatase superfamily, in which the native reaction for one member of the superfamily is often a promiscuous side reaction for another. Moreover, despite the similarity of the substrates involved, the reactions catalysed (cleavage of P-O and S-O bonds) proceed with very different solvation and protonation requirements. We present here a detailed study of the promiscuous catalytic activity of three evolutionarily related but structurally different members of this superfamily: an arylsulfatase from Pseudonomas aeruginosa (PAS) and two phosphonate monoester hydrolases (PMH) from Rhizobium leguminosarum and Burkholderia caryophylli. The latter enzymes are particularly interesting due to the fact that, in addition to their broad promiscuity, they are also capable of catalysing a xenobiotic substrate for which no natural catalyst is known. We demonstrate that subtle changes in active site residues can have a significant impact on mechanism. However, despite such mechanistic variation, the promiscuity simply arises out of the ability of the non-native substrates to exploit the pre-existing electrostatic pre-organization of the active site towards the native substrate. We believe this provides an example of chemically-driven protein evolution within an enzyme superfamily.