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

Christopher D Bayer,Florian Hollfelder,Bert Van Loo

doi:10.1002/cbic.201600657

Christopher D Bayer, Florian Hollfelder + Show 1 more

Open Access

https://doi.org/10.1002/cbic.201600657

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Journal: ChemBioChem	Publication Date: May 2, 2017
Citations: 17	License type: CC BY 4.0

Affiliation: University of Cambridge, University of Münster

Abstract

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.

Highlights

In apparent conflict with traditional views on enzymatic catalysis, promiscuous enzymes are not exclusively specific for single substrates, but turn over multiple substrates, even if those compounds differ substantially in their molecular recognition properties.[1]
We investigated the accessibility of improvements of promiscuous activities by single amino acid replacement mutagenesis of an analogous position (HisA in Figure 1 B) in one representative each of the AS (SpAS1) and phosphonate monoester hydrolase (PMH) (RlPMH) families
This residue is located within hydrogen-bonding distance of the fGly nucleophile,[11,14,17,34] and the nature of this residue can be used to differentiate between ASs (His) and PMHs (Asp/Thr)

Summary

Introduction

In apparent conflict with traditional views on enzymatic catalysis, promiscuous enzymes are not exclusively specific for single substrates, but turn over multiple substrates, even if those compounds differ substantially in their molecular recognition properties.[1]. The substrates hydrolyzed by members of the AP superfamily are diverse in their molecular recognition properties: members of this superfamily have been shown to convert substrates with one, two, or no negative charges, and the substrates have half-lives from several months to millions of years,[2,6,11,12,13,14,15,16,17,18] implying very different catalytic requirements This chemical diversity is further supported by the observation that the hydrolytic reactions of phosphate diesters and phosphonate monoesters proceed via concerted TSs,[36,37] whereas those of phosphate and sulfate monoesters are characterized by more expanded, dissociative TSs.[37,38,39,40]. Kinetic data for mutants were measured, and the data were subjected to statistical analyses to study relationships between mutations and the observed rates as a function of chemical properties of substrates or reaction types (e.g., substrate charge, nature of TSs)

Results and Discussion

Conclusions

Conflict of Interest