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Specificity Effects of Amino Acid Substitutions in Promiscuous Hydrolases: Context-Dependence of Catalytic Residue Contributions to Local Fitness Landscapes in Nearby Sequence Space

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Bayer, CD 
van Loo, B 
Hollfelder, Florian  ORCID logo


Catalytic promiscuity can facilitate evolution of enzyme functions-a multifunctional catalyst may act as a springboard for efficient functional adaptation. We test the effect of single mutations on multiple activities in two groups of promiscuous AP superfamily members to probe this hypothesis. We quantify the effect of site-saturating mutagenesis of an analogous, nucleophile-flanking residue in two superfamily members: an arylsulfatase (AS) and a phosphonate monoester hydrolase (PMH). Statistical analysis suggests that no one physicochemical characteristic alone explains the mutational effects. Instead, these effects appear to be dominated by their structural context. Likewise, the effect of changing the catalytic nucleophile itself is not reaction-type-specific. Mapping of "fitness landscapes" of four activities onto the possible variation of a chosen sequence position revealed tremendous potential for respecialization of AP superfamily members through single-point mutations, highlighting catalytic promiscuity as a powerful predictor of adaptive potential.



catalytic promiscuity, fitness landscapes, molecular evolution, mutagenesis, phosphate transfer

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Biotechnology and Biological Sciences Research Council (BB/I004327/1)
European Research Council (208813)
This research was funded by the Biological and Biotechnological Research Council (BBSRC) and the Human Frontiers Science Programme. C.D.B. was supported by a BBSRC studentship and the Cambridge European Trust.