Mechanistic Elucidation and Stereochemical Consequences of Alternative Binding of Alkenyl Substrates by Engineered Arylmalonate Decarboxylase
(2025) In Journal of the American Chemical Society 147(43). p.39271-39283- Abstract
The cofactor-free arylmalonate decarboxylase (AMDase) is a valuable biocatalyst for synthesizing α-aryl and α-alkenyl alkanoic acids with excellent stereoselectivity. We engineered a new hydrophobic pocket in (S)-selective AMDase mutants, creating AMDase ICPLLG with enhanced activity. For the investigation of the mechanism, we synthesized isotope-labeled, pseudochiral 2-methyl-2-vinyl malonate via an auxiliary-based asymmetric route using a chiral imidazolidinone to enable stereoselective bis-alkylation of malonates. Our results reveal striking substrate-dependent stereochemical behavior: AMDase ICPLLG decarboxylates prochiral aromatic malonates with retention of configuration at the α-carbon. The critical Cys residue adds a proton from... (More)
The cofactor-free arylmalonate decarboxylase (AMDase) is a valuable biocatalyst for synthesizing α-aryl and α-alkenyl alkanoic acids with excellent stereoselectivity. We engineered a new hydrophobic pocket in (S)-selective AMDase mutants, creating AMDase ICPLLG with enhanced activity. For the investigation of the mechanism, we synthesized isotope-labeled, pseudochiral 2-methyl-2-vinyl malonate via an auxiliary-based asymmetric route using a chiral imidazolidinone to enable stereoselective bis-alkylation of malonates. Our results reveal striking substrate-dependent stereochemical behavior: AMDase ICPLLG decarboxylates prochiral aromatic malonates with retention of configuration at the α-carbon. The critical Cys residue adds a proton from the same face of the substrate as the leaving carboxylate. Interestingly, the same mutant decarboxylates the corresponding alkenyl malonate with inversion of configuration, i.e., with protonation from the opposite face. Kinetic isotope effect measurements and QM/MM metadynamics calculations suggest that alkenyl malonates adopt an alternative binding mode and undergo decarboxylation via a borderline concerted mechanism instead of a stepwise mechanism. This new pathway changes the stereochemical preference. We exploited this strategy to decarboxylate sterically hindered alkenyl malonates (substrates not converted by wild-type AMDase) with high stereoselectivity. The engineered hydrophobic pocket in (S)-selective AMDase mutants expands the substrate scope for synthesizing enantiomerically pure α-aryl and α-alkenyl butanoic acids. This work demonstrates a new approach (a mechanistic change) to engineer the substrate range and stereoselectivity of enzymes.
(Less)
- author
- van der Pol, Elske
; Schlatzer, Thomas
; Hoffka, Gyula
LU
; Di Geronimo, Bruno
; Eder, Johannes
; Schweiger, Anna K.
; Karava, Marianna
; Gross, Dominik
; Fischer, Roland C.
; Kracher, Daniel
; Kazlauskas, Romas
; Miyamoto, Kenji
; Kamerlin, Shina Caroline Lynn
LU
; Breinbauer, Rolf
and Kourist, Robert
(Less) - organization
- publishing date
- 2025-10-29
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Journal of the American Chemical Society
- volume
- 147
- issue
- 43
- pages
- 13 pages
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- pmid:41085469
- scopus:105020375243
- ISSN
- 0002-7863
- DOI
- 10.1021/jacs.5c10721
- language
- English
- LU publication?
- yes
- additional info
- Publisher Copyright: © 2025 The Authors. Published by American Chemical Society
- id
- 10cc2e32-e851-4e67-be78-42a17c40524e
- date added to LUP
- 2025-12-17 10:52:51
- date last changed
- 2025-12-18 03:54:44
@article{10cc2e32-e851-4e67-be78-42a17c40524e,
abstract = {{<p>The cofactor-free arylmalonate decarboxylase (AMDase) is a valuable biocatalyst for synthesizing α-aryl and α-alkenyl alkanoic acids with excellent stereoselectivity. We engineered a new hydrophobic pocket in (S)-selective AMDase mutants, creating AMDase ICPLLG with enhanced activity. For the investigation of the mechanism, we synthesized isotope-labeled, pseudochiral 2-methyl-2-vinyl malonate via an auxiliary-based asymmetric route using a chiral imidazolidinone to enable stereoselective bis-alkylation of malonates. Our results reveal striking substrate-dependent stereochemical behavior: AMDase ICPLLG decarboxylates prochiral aromatic malonates with retention of configuration at the α-carbon. The critical Cys residue adds a proton from the same face of the substrate as the leaving carboxylate. Interestingly, the same mutant decarboxylates the corresponding alkenyl malonate with inversion of configuration, i.e., with protonation from the opposite face. Kinetic isotope effect measurements and QM/MM metadynamics calculations suggest that alkenyl malonates adopt an alternative binding mode and undergo decarboxylation via a borderline concerted mechanism instead of a stepwise mechanism. This new pathway changes the stereochemical preference. We exploited this strategy to decarboxylate sterically hindered alkenyl malonates (substrates not converted by wild-type AMDase) with high stereoselectivity. The engineered hydrophobic pocket in (S)-selective AMDase mutants expands the substrate scope for synthesizing enantiomerically pure α-aryl and α-alkenyl butanoic acids. This work demonstrates a new approach (a mechanistic change) to engineer the substrate range and stereoselectivity of enzymes.</p>}},
author = {{van der Pol, Elske and Schlatzer, Thomas and Hoffka, Gyula and Di Geronimo, Bruno and Eder, Johannes and Schweiger, Anna K. and Karava, Marianna and Gross, Dominik and Fischer, Roland C. and Kracher, Daniel and Kazlauskas, Romas and Miyamoto, Kenji and Kamerlin, Shina Caroline Lynn and Breinbauer, Rolf and Kourist, Robert}},
issn = {{0002-7863}},
language = {{eng}},
month = {{10}},
number = {{43}},
pages = {{39271--39283}},
publisher = {{The American Chemical Society (ACS)}},
series = {{Journal of the American Chemical Society}},
title = {{Mechanistic Elucidation and Stereochemical Consequences of Alternative Binding of Alkenyl Substrates by Engineered Arylmalonate Decarboxylase}},
url = {{http://dx.doi.org/10.1021/jacs.5c10721}},
doi = {{10.1021/jacs.5c10721}},
volume = {{147}},
year = {{2025}},
}