University of Groningen
Biocatalytic asymmetric hydroamination by native and engineered carbon-nitrogen lyases Zhang, Jielin
DOI:
10.33612/diss.93007154
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Publication date: 2019
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Zhang, J. (2019). Biocatalytic asymmetric hydroamination by native and engineered carbon-nitrogen lyases: new enzymes to prepare amino acid precursors to pharmaceuticals and food additives. University of Groningen. https://doi.org/10.33612/diss.93007154
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Biocatalytic asymmetric hydroamination by native
and engineered carbon-nitrogen lyases
New enzymes to prepare amino acid precursors to pharmaceuticals
and food additives
Jielin Zhang
The research described in this thesis was carried out in the Department of Chemical and Pharmaceutical Biology (Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands) and was financially supported by the China Scholarship Council, the Netherlands Organization of Scientific Research (VICI grant 724.016.002, ECHO grant 700.59.042), the European Union 7th framework project Metaexplore (KBBE-2007-3-3-05, grant agreement number 222625), and the European Research Council (Starting grant 242293, PoC grant 713483).
The research work was carried out according to the requirements of the Graduate School of Science, Faculty of Science and Engineering, University of Groningen, The Netherlands.
Printing of this thesis was financially supported by the University Library and the Graduate School of Science, Faculty of Science and Engineering, University of Groningen, The Netherlands.
ISBN: 978-94-034-1845-2 (printed version) ISBN: 978-94-034-1844-5 (electronic version) Printing: Ridderprint BV, www.ridderprint.nl Layout and Cover design: Jielin Zhang
Cover picture: Reflection by Qingzhen Ma & Hui Zhang
Copyright © 2019 Jielin Zhang. All rights are reserved. No part of this thesis may be reproduced or transmitted in any form or by any means without the prior permission in wiritng of the author.
Biocatalytic asymmetric hydroamination by
native and engineered carbon-nitrogen lyases
New enzymes to prepare amino acid precursors to
pharmaceuticals and food additives
PhD thesis
to obtain the degree of PhD at the University of Groningen
on the authority of the Rector Magnificus Prof. E. Sterken
and in accordance with
the decision by the College of Deans. This thesis will be defended in public on
Monday 26 August 2019 at 11.00 hours
by
Jielin Zhang
born on 22 June 1988 in Heibei, ChinaSupervisors
Prof. G.J. Poelarends Prof. W.J. Quax
Assessment committee Prof. W.J.H. van Berkel Prof. M.J.E.C. van der Maarel Prof. M.W. Fraaije
“We take a handful of sand from the endless landscape of awareness around us and call that handful of sand the world.”
Robert M. Pirsig
Zen and the Art of Motorcycle Maintenance
Paranimfen
Lieuwe Biewenga
Table of Contents
Aim and Outline of This Thesis
9Chapter 1
Introduction13
Chapter 2
Chemoenzymatic Asymmetric Synthesis of the Metallo-β-Lactamase Inhibitor Aspergillomarasmine A and Related Aminocarboxylic Acids
27
Chapter 3
Structural Basis for the Catalytic Mechanism of Ethylenediamine-N,N′-Disuccinic Acid Lyase, a Carbon-Nitrogen Bond-Forming Enzyme with Broad Substrate Scope
75
Chapter 4
Biocatalytic Enantioselective Hydroaminations for Production of N-Cycloalkyl-Substituted L-Aspartic Acids Using Two C-N Lyases
105
Chapter 5
Enantioselective Synthesis of Chiral Synthons for Artificial Dipeptide Sweeteners Catalyzed by an Engineered C-N Lyase
137
Chapter 6
Modular Enzymatic Cascade Synthesis of Vitamin B5 and its Derivatives
175
Chapter 7
Summary and Future Perspectives
213
Nederlandse Samenvatting
221Acknowledgement
225Aim and outline of this thesis
9
Aim and outline of this thesis
L-aspartic acid derivatives are unnatural amino acids with a broad range of applications in neurobiological research and the synthesis of pharma- and nutraceuticals. Although carbon-nitrogen bond-forming C-N lyases are attractive enzymes to prepare such compounds, only two C-N lyases known as aspartate ammonia lyase (aspartase) and 3-methylaspartate ammonia lyase (MAL) have been carefully explored for their usefulness in the synthesis of difficult L-aspartic acid derivatives. Unfortunately, the substrate scope of these enzymes is rather limited, with low or no reactivity for desired non-native substrates. Expanding the toolbox of C-N lyases for amino acid synthesis by enzyme discovery and engineering is thus highly interesting. Another field with great potential is the use of C-N lyases in multienzymatic and chemoenzymatic cascades, allowing the construction of artificial metabolic pathways for the more sustainable and step-economic synthesis of complex amino acid molecules starting from simple and cheap building blocks.
The work described in this thesis aimed to expand the biocatalytic applications of C-N lyases for asymmetric synthesis of important amino acid precursors to biologically active compounds and food additives. For this, the MAL enzyme and a newly identified ethylenediamine‑N,N’‑disuccinic acid lyase (EDDS lyase), as well as engineered variants of both these C-N lyases, were investigated for their usefulness in the selective (cascade) synthesis of difficult aminocarboxylic acid products.
Chapter 1 gives a brief overview of the properties of EDDS lyase and MAL, including their biochemical, structural and mechanistic features and biocatalytic applications to prepare unnatural amino acids.
In Chapter 2, a chemoenzymatic methodology for asymmetric synthesis of the fungal natural products aspergillomarasmine A (AMA), aspergillomarasmine B (AMB), toxin A and related aminocarboxylic acids is reported. AMA is a potent inhibitor of metallo-β-lactamases, with great pharmaceutical potential in battling bacterial resistance to β-lactam antibiotics. This step-economic (chemo)enzymatic route towards AMA, AMB, and related aminocarboxylic acids highlights a highly regio- and stereoselective C-N bond forming step catalyzed by EDDS lyase.
Our knowledge of EDDS lyase was broadened by the identification and structural characterization of EDDS lyase from Chelativorans sp. BNC1 (Chapter 3). The determined crystal structures of EDDS lyase in unliganded and substrate- and product-bound forms not only support a general base-catalyzed deamination mechanism characteristic for members of the aspartase/fumarase superfamily, but also provide structural basis for future enzyme engineering.
Aim and outline of this thesis
10
The potential for biocatalytic application of EDDS lyase and MAL-Q73A was further demonstrated by the enantioselective synthesis of N-substituted L-aspartic acid derivatives with diverse homo- and heterocycloalkyl substituents (Chapter 4). Another example is given in Chapter 5, which describes the engineering of an improved EDDS lyase variant for efficient enantioselective production of N-(3,3-dimethylbutyl)-L-aspartic acid and N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-L-aspartic acid, important precursors to the artificial dipeptide sweeteners neotame and advantame, respectively.
Chapter 6 describes the development of a one-pot three-step enzymatic cascade for stereoselective synthesis of vitamin B5 [(R)-pantothenic acid] and both diastereoisomers of
α-methyl-substituted vitamin B5, which are valuable precursors to promising antimicrobials
against Plasmodium falciparum and multidrug-resistant Staphylococcus aureus, using a C-N lyase (MAL), an appropriate decarboxylase, and pantothenate synthetase enzymes.
Lastly, Chapter 7 provides a summary of the work presented in this thesis, concluding remarks, and some perspectives for future research.
