Designing artificial enzymes with unnatural amino acids

Enzymen zijn krachtige katalysatoren die een centrale rol spelen in alle biologische reacties in de natuur. Ze staan bekend om het katalyseren van reacties met hoge activiteit en opmerkelijke selectiviteit, maar ze zijn gevoelig voor de het milieu waarin ze zich bevinden en accepteren slechts een gelimiteerd aantal substraten en reacties. Vandaar dat er veel werk is verricht om een alternatief voor natuurlijke enzymen te ontwikkelen, de zogenaamde kunstmatige enzymen. Deze enzymen kunnen reacties uitvoeren met dezelfde efficiëntie, maar hebben waardevolle voordelen ten opzichte van de natuurlijke systemen, zoals de mogelijkheid om onnatuurlijke reacties uit te voeren. Dit is van groot praktisch belang, en heeft potentie voor toepassingen in industriële processen. Dit proefschrift beschrijft de ontwikkeling van een nieuwe methode om kunstmatige enzymen te creëren door gebruik te maken van genetisch gecodeerde onnatuurlijke aminozuren als metaalbindende groep, of katalytisch residu. Onnatuurlijke aminozuren bevatten een groot scala aan zijgroepen en functionele groepen en bieden daardoor veel mogelijkheden om het repertoire van kunstmatige enzymen uit te breiden. Een groot deel van dit proefschrift beschrijft de toepassing van deze methode voor de creatie van nieuwe kunstmatige metaalenzymen, via het inbouwen van de niet-proteogene metaalbindende aminozuren (2,2΄-bipyridin-5-yl)alanine en (8-hydroxyquinolin-3-yl)aniline in de structuur van LmrR. Deze nieuwe metaalenzymen waren succesvol in de katalyse van uitdagende reacties, zoals de Friedel-Crafts alkyleringsreactie en de enantioselectieve geconjugeerde additie van water. Ook toonden ze veelbelovende activiteit voor andere reacties. Daarnaast werd het niet-metaalbindende p-aminofenylalanine gebruikt als katalytisch residu voor een nieuwe klasse enzymen. Met behulp van de nucleofiliciteit van aniline in de zijketen van dit onnatuurlijke aminozuur werd een kunstmatig enzym bereid dat de vorming van hydrazonen kan katalyseren. Hiermee vertegenwoordigt dit enzym een nieuw type katalysatoren voor een reactie die niet in de natuur voorkomt

Unlocking Iminium Catalysis in Artificial Enzymes to Create a Friedel-Crafts Alkylase

[Image: see text] The construction and engineering of artificial enzymes consisting of abiological catalytic moieties incorporated into protein scaffolds is a promising strategy to realize non-natural mechanisms in biocatalysis. Here, we show that incorporation of the noncanonical amino acid para-aminophenylalanine (pAF) into the nonenzymatic protein scaffold LmrR creates a proficient and stereoselective artificial enzyme (LmrR_pAF) for the vinylogous Friedel–Crafts alkylation between α,β-unsaturated aldehydes and indoles. pAF acts as a catalytic residue, activating enal substrates toward conjugate addition via the formation of intermediate iminium ion species, while the protein scaffold provides rate acceleration and stereoinduction. Improved LmrR_pAF variants were identified by low-throughput directed evolution advised by alanine-scanning to obtain a triple mutant that provided higher yields and enantioselectivities for a range of aliphatic enals and substituted indoles. Analysis of Michaelis–Menten kinetics of LmrR_pAF and evolved mutants reveals that different activities emerge via evolutionary pathways that diverge from one another and specialize catalytic reactivity. Translating this iminium-based catalytic mechanism into an enzymatic context will enable many more biocatalytic transformations inspired by organocatalysis

A Hydroxyquinoline-Based Unnatural Amino Acid for the Design of Novel Artificial Metalloenzymes

We have examined the potential of the noncanonical amino acid (8-hydroxyquinolin-3-yl)alanine (HQAla) for the design of artificial metalloenzymes. HQAla, a versatile chelator of late transition metals, was introduced into the lactococcal multidrug-resistance regulator (LmrR) by stop codon suppression methodology. LmrR_HQAla was shown to complex efficiently with three different metal ions, Cu-II, Zn(II)and Rh(III)to form unique artificial metalloenzymes. The catalytic potential of the Cu-II-bound LmrR_HQAla enzyme was shown through its ability to catalyse asymmetric Friedel-Craft alkylation and water addition, whereas the Zn-II-coupled enzyme was shown to mimic natural Zn hydrolase activity

Artificial Metalloproteins for Binding and Stabilization of a Semiquinone Radical

The interaction of a number of first-row transition-metal ions with a 2,2'-bipyridyl alanine (bpyA) unit incorporated into the lactococcal multidrug resistance regulator (LmrR) scaffold is reported. The composition of the active site is shown to influence binding affinities. In the case of Fe(II), we demonstrate the need of additional ligating residues, in particular those containing carboxylate groups, in the vicinity of the binding site. Moreover, stabilization of di-tert-butylsemiquinone radical (DTB-SQ) in water was achieved by binding to the designed metalloproteins, which resulted in the radical being shielded from the aqueous environment. This allowed the first characterization of the radical semiquinone in water by resonance Raman spectroscopy

Design of Artificial Enzymes: Insights into Protein Scaffolds

The design of artificial enzymes has emerged as a promising tool for the generation of potent biocatalysts able to promote new-to-nature reactions with improved catalytic performances, providing a powerful platform for wide-ranging applications and a better understanding of protein functions and structures. The selection of an appropriate protein scaffold plays a key role in the design process. This review aims to give a general overview of the most common protein scaffolds that can be exploited for the generation of artificial enzymes. Several examples are discussed and categorized according to the strategy used for the design of the artificial biocatalyst, namely the functionalization of natural enzymes, the creation of a new catalytic site in a protein scaffold bearing a wide hydrophobic pocket and de novo protein design. The review is concluded by a comparison of these different methods and by our perspective on the topic

Non-canonical amino acids as a tool for the thermal stabilization of enzymes

Biocatalysis has become a powerful alternative for green chemistry. Expanding the range of amino acids used in protein biosynthesis can improve industrially appealing properties such as enantioselectivity, activity and stability. This review will specifically delve into the thermal stability improvements that non-canonical amino acids (ncAAs) can confer to enzymes. Methods to achieve this end, such as the use of halogenated ncAAs, selective immobilization and rational design, will be discussed. Additionally, specific enzyme design considerations using ncAAs are discussed along with the benefits and limitations of the various approaches available to enhance the thermal stability of enzymes

Expanding the Genetic Code:Incorporation of Functional Secondary Amines via Stop Codon Suppression

Enzymes are attractive catalysts for chemical industries, and their use has become a mature alternative to conventional chemical methods. However, biocatalytic approaches are often restricted to metabolic and less complex reactivities, given the limited amount of functional groups present. This drawback can be addressed by incorporating non-canonical amino acids (ncAAs) harboring new-to-nature chemical groups. Inspired by organocatalysis, we report the design, synthesis and characterization of a panel of ncAAs harboring functional secondary amines and their cellular incorporation into different protein scaffolds. D/L-pyrrolidine- and D/L-piperidine-based ncAAs were successfully site-specifically incorporated into proteins via stop codon suppression methodology. To demonstrate the utility of these ncAAs, the catalytic performance of the obtained artificial enzymes was investigated in a model Michael addition reaction. The incorporation of pyrrolidine- and piperidine- based ncAAs significantly expands the available toolbox for protein engineering and chemical biology applications.</p

A designer enzyme for hydrazone and oxime formation featuring an unnatural catalytic aniline residue

Creating designer enzymes with the ability to catalyse abiological transformations is a formidable challenge. Efforts toward this goal typically consider only canonical amino acids in the initial design process. However, incorporating unnatural amino acids that feature uniquely reactive side chains could significantly expand the catalytic repertoire of designer enzymes. To explore the potential of such artificial building blocks for enzyme design, here we selected p-aminophenylalanine as a potentially novel catalytic residue. We demonstrate that the catalytic activity of the aniline side chain for hydrazone and oxime formation reactions is increased by embedding p-aminophenylalanine into the hydrophobic pore of the multidrug transcriptional regulator from Lactococcus lactis. Both the recruitment of reactants by the promiscuous binding pocket and a judiciously placed aniline that functions as a catalytic residue contribute to the success of the identified artificial enzyme. We anticipate that our design strategy will prove rewarding to significantly expand the catalytic repertoire of designer enzymes in the future