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

An Artificial Heme Enzyme for Cyclopropanation Reactions

An artificial heme enzyme was created through self-assembly from hemin and the lactococcal multidrug resistance regulator (LmrR). The crystal structure shows the heme bound inside the hydrophobic pore of the protein, where it appears inaccessible for substrates. However, good catalytic activity and moderate enantioselectivity was observed in an abiological cyclopropanation reaction. We propose that the dynamic nature of the structure of the LmrR protein is key to the observed activity. This was supported by molecular dynamics simulations, which showed transient formation of opened conformations that allow the binding of substrates and the formation of pre-catalytic structures

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

Artificial Imine Reductases: Developments and Future Directions

Biocatalytic imine reduction has been a topic of intense research by the artificial metalloenzyme community in recent years. Artificial constructs, together with natural enzymes, have been engineered to produce chiral amines with high enantioselectivity. This review examines the design of the main classes of artificial imine reductases reported thus far and summarises approaches to enhancing their catalytic performance using complementary methods. Examples of utilising these biocatalysts in vivo or in multi-enzyme cascades have demonstrated the potential that artIREDs can offer, however, at this time their use in biocatalysis remains limited. This review explores the current scope of artIREDs and the strategies used for catalyst improvement, and examines the potential for artIREDs in the future

Latest Developments in Metalloenzyme Design and Repurposing

In the past decade, artificial metalloenzymes (AMEs) have emerged as attractive alternatives to more traditional homogeneous catalysts and enzymes. This microreview presents a selection of recent achievements in the design of such hybrid catalysts. These include artificial zinc hydrolases and metathesases, the heme-protein repurposing for C–H, N–H, and S–H insertion reactions, novel light-driven redox hybrid catalysts, novel scaffold proteins, and metallocofactor anchoring techniques and metalloenzyme models