Ancestral reconstruction and experimental testing of long- and short term evolvability using the AP-superfamily as a model system

Eenink B, Heberlein M, Mair P, Schüürmann J, Dilkaute C, Jose J, Hollfelder F, Bornberg-Bauer E, van Loo B

Abstract

How functional proteins combine thecontinuous exploration of nearbysequence space for novel functionswhile maintaining their existingfunctions is key to understanding theevolution of new functions. In essencewe want to understand how proteins areaffected by mutation and to use thatknowledge to provide guidelines on howto efficiently create artificial systemswith novel functionalities. The currentdayvariety of proteins within proteinsuperfamilies has evolved out ofcommon ancestors that are evolvable bydefinition. Characterization of theseancestral states can therefore provideinsight into which features are typical ofan evolvable protein. Furthermore theseancestral states can possibly be used asa starting point for directed evolutiontoward completely novel functions.Initially we are focusing on thedivergence between arylsulfatases (ASs)and phosphonate monoester hydrolases(PMHs)1-4 in the alkaline phosphatase(AP) superfamily.Four ancestral nodes of the AS cladehave so far been characterized withregards to substrate specificity andthermostability.Five extant AS's and five ancestral nodesof the AS clade have succesfully beenexpressed on the surface of E. coli, andconditions for the autodisplay havebeen fine-tuned.