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Broadening the Scope of Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology

English title Broadening the Scope of Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology
Applicant Ward Thomas R.
Number 162348
Funding scheme Project funding
Research institution Institut für Anorganische Chemie Universität Basel
Institution of higher education University of Basel - BS
Main discipline Inorganic Chemistry
Start/End 01.10.2015 - 30.09.2018
Approved amount 620'000.00
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All Disciplines (2)

Discipline
Inorganic Chemistry
Organic Chemistry

Keywords (7)

Artificial metalloenzymes; Cross-coupling; Organometallic chemistry; Chemogenetic optimization; Alcane hydroxylation; Protein engineering; biotin-streptavidin technology

Lay Summary (French)

Lead
Ce projet de recherche vise à développer des solutions catalytiques alternatives pour résoudre des problèmes environmentaux urgents
Lay summary

La catalyse est un procédé indispensable à l’industrie pharmaceutique car elle permet de réduire les coûts et les impacts environnementaux de son activité chimique. Le catalyseur est une substance (bio)chimique qui permet d’accélérer la cinétique de formation des produits lorsqu’il est ajouté à une réaction chimique. Des composés organométalliques (appelés catalyseurs homogènes) ainsi que des protéines (des enzymes) ont démontré leur utilité en tant que catalyseurs. Ils comportent tous deux des avantages et des désavantages.

Dans le projet tel qu’il a été soumis, les avantages de ces deux types de catalyseurs sont combinés afin de créer un nouveau type de catalyseurs appelés les métalloenzymes artificielles. Ces catalyseurs hybrides résultent de l’incorporation d’un composé organométallique à une protéine. Au cours de la dernière décennie, les métalloenzymes artificielles se sont en effet révélées être des catalyseurs aux propriétés catalytiques surprenantes et complémentaires aux systèmes plus traditionnels des enzymes et des catalyseurs homogènes.

Le projet proposé vise à exploiter la streptavidine comme protéine hôte pour divers catalyseurs organométalliques afin de créer de nouvelles métalloenzymes artificielles qui seront capable d’être des catalyseurs pour les transformations chimiques les plus difficiles à réaliser telles la réaction de couplage carbone-carbone et l’activation des liaisons C–H réputées inertes. Afin d’atteindre cet objectif avant-gardiste, des stratégies d’optimisation chimique et génétique seront exploitées.

Ce projet vise donc ces deux buts principaux : i) offrir des alternatives catalytiques efficientes pour résoudre des problèmes environnementaux urgents (réduire le gaz à effet de serre), ii) comprendre et exploiter les interactions faibles entre la protéine (métalloenzyme artificielle) et le substrat (produit de départ) afin de contrôler de manière spécifique et précise les cycles catalytiques pour lesquels il n’existe à ce jour pas de solution satisfaisante.

 

Direct link to Lay Summary Last update: 04.10.2015

Responsible applicant and co-applicants

Employees

Publications

Collaboration

Group / person Country
Types of collaboration
Prof. Sven Panke, DBSSE, ETHZ at Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Prof. David Baker, Univ. Washington Seattle United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Awards

Title Year
Ambizione Fellowship 2018
Marie Curie Fellowship 2017
RSC award in Bioinorganic Chemistry 2017

Associated projects

Number Title Start Funding scheme
182046 Directed Evolution of Artificial Metalloproteins and Metalloenzymes 01.10.2018 Project funding
144354 Directed Evolution of Artificial Metalloenzymes : Towards Chemical Biology Applications 01.10.2012 Project funding

Abstract

In the past decade, artificial metalloenzymes (ArMs) have emerged as attractractive alternatives to homogeneous catalysts and enzymes. Such hybrid catalysts result from incorporation of an organometallic catalyst precursor within a macromolecule (protein or DNA) and display features reminiscent of both enzymes (genetic engineering, precise control of second coordination sphere interactions etc.) and homogeenous catalysts (broad reaction repertoire and substrate scope etc.)Relying on either covalent-, dative- or supramolecular anchoring strategies of the abiotic cofactor within a macromolecular host, over twenty ArMs-catalysed transformations have been reported to date. In this context, the biotin-streptavidin technology has proven remarkably versatile for the implementation of various ArMs. The following features of the biotin-streptavidn technology are particularly attractive: i) Streptavidin (Sav) can be recombinantly produced in E. coli (up to 9 g ·L-1 soluble and functional protein); ii) The association constant of biotinylated cofactors with Sav is very high (typically Ka > 10^10 M-1), thus ensuring quantitative localization of the biotinylated catalyst within Sav even in complex media; iii) Sav is remarkably stable towards various chaotropic agents (pH, solvents, temperature etc.) as well as genetic modifications; iv) The size of the biotin-binding vestibule is ideally suited to accommodate both the metal cofactor and its substrate(s). However, X-ray structural analysis of ArMs based on the biotin-streptavidin technology reveals that the biotinylated metal moiety is partially exposed to the reaction medium.The aim of this proposal is to genetically engineer Sav to tailor the ArM’s active site for specific catalytic needs. For this purpose, we will rely on the introduction of multiple mutations and/or of new structural motifs (e.g. fusion proteins) on the Sav gene. Preliminary experiments demonstrate that Sav is remarkably tolerant towards introduction of additional (GGS)n stretches (n = 5) around the active site. We anticipate that embedding the metal moiety within a deeper active site will offer better opportunities to fine-tune the activity and the selectivity of the resulting ArMs. To benchmark the engineered biotin-binding fusion proteins, two ArMs-catalyzed reactions will be developed: a palladium-catalyzed Suzuki cross-coupling reaction as well as an iron catalyzed alkane hydroxylation reaction. For the former reaction, we anticipate that shielding the biotinylated cofactor will protect palladium towards poisonous cellular components, eventually allowing the reaction to be performed in vivo under truly catalytic conditions. In a biomimetic spirit, anchoring a highly active Fe=O moiety within a hydrophobic and well-shielded environment will allow to selectively hydroxylate alkanes and prevent the over-oxidation of the resulting alcohol, ultimately leading to the development of an artificial methane monooxygenase.
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