Skip to main content

Lund University Publications

LUND UNIVERSITY LIBRARIES

Evolutionary repurposing of a sulfatase : A new Michaelis complex leads to efficient transition state charge offset

Miton, Charlotte M ; Jonas, Stefanie ; Fischer, Gerhard ; Duarte, Fernanda ; Mohamed, Mark F ; van Loo, Bert ; Kintses, Bálint ; Kamerlin, Shina C L LU orcid ; Tokuriki, Nobuhiko and Hyvönen, Marko , et al. (2018) In Proceedings of the National Academy of Sciences of the United States of America 115(31). p.7293-7302
Abstract

The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono- and... (More)

The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono- and diester hydrolyses were only marginally affected (≤50-fold). Structural, kinetic, and in silico characterizations of the evolutionary intermediates revealed that two key mutations, T50A and M72V, locally reshaped the active site, improving access to the catalytic machinery for the phosphonate. Measured transition state (TS) charge changes along the trajectory suggest the creation of a new Michaelis complex (E•S, enzyme-substrate), with enhanced leaving group stabilization in the TS for the promiscuous phosphonate (βleavinggroup from -1.08 to -0.42). Rather than altering the catalytic machinery, evolutionary repurposing was achieved by fine-tuning the molecular recognition of the phosphonate in the Michaelis complex, and by extension, also in the TS. This molecular scenario constitutes a mechanistic alternative to adaptation solely based on enzyme flexibility and conformational selection. Instead, rapid functional transitions between distinct chemical reactions rely on the high reactivity of permissive active-site architectures that allow multiple substrate binding modes.

(Less)
Please use this url to cite or link to this publication:
author
; ; ; ; ; ; ; ; and , et al. (More)
; ; ; ; ; ; ; ; ; and (Less)
publishing date
type
Contribution to journal
publication status
published
keywords
Arylsulfatases/chemistry, Catalysis, Catalytic Domain, Directed Molecular Evolution, Hydrolysis, Organophosphorus Compounds/chemistry, Protein Conformation
in
Proceedings of the National Academy of Sciences of the United States of America
volume
115
issue
31
pages
7293 - 7302
publisher
National Academy of Sciences
external identifiers
  • scopus:85051788064
  • pmid:30012610
ISSN
1091-6490
DOI
10.1073/pnas.1607817115
language
English
LU publication?
no
id
46153840-e6a5-4d11-a848-e4c7478310de
date added to LUP
2025-01-11 21:05:03
date last changed
2025-03-09 08:11:40
@article{46153840-e6a5-4d11-a848-e4c7478310de,
  abstract     = {{<p>The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono- and diester hydrolyses were only marginally affected (≤50-fold). Structural, kinetic, and in silico characterizations of the evolutionary intermediates revealed that two key mutations, T50A and M72V, locally reshaped the active site, improving access to the catalytic machinery for the phosphonate. Measured transition state (TS) charge changes along the trajectory suggest the creation of a new Michaelis complex (E•S, enzyme-substrate), with enhanced leaving group stabilization in the TS for the promiscuous phosphonate (βleavinggroup from -1.08 to -0.42). Rather than altering the catalytic machinery, evolutionary repurposing was achieved by fine-tuning the molecular recognition of the phosphonate in the Michaelis complex, and by extension, also in the TS. This molecular scenario constitutes a mechanistic alternative to adaptation solely based on enzyme flexibility and conformational selection. Instead, rapid functional transitions between distinct chemical reactions rely on the high reactivity of permissive active-site architectures that allow multiple substrate binding modes.</p>}},
  author       = {{Miton, Charlotte M and Jonas, Stefanie and Fischer, Gerhard and Duarte, Fernanda and Mohamed, Mark F and van Loo, Bert and Kintses, Bálint and Kamerlin, Shina C L and Tokuriki, Nobuhiko and Hyvönen, Marko and Hollfelder, Florian}},
  issn         = {{1091-6490}},
  keywords     = {{Arylsulfatases/chemistry; Catalysis; Catalytic Domain; Directed Molecular Evolution; Hydrolysis; Organophosphorus Compounds/chemistry; Protein Conformation}},
  language     = {{eng}},
  month        = {{07}},
  number       = {{31}},
  pages        = {{7293--7302}},
  publisher    = {{National Academy of Sciences}},
  series       = {{Proceedings of the National Academy of Sciences of the United States of America}},
  title        = {{Evolutionary repurposing of a sulfatase : A new Michaelis complex leads to efficient transition state charge offset}},
  url          = {{http://dx.doi.org/10.1073/pnas.1607817115}},
  doi          = {{10.1073/pnas.1607817115}},
  volume       = {{115}},
  year         = {{2018}},
}