Design and evolution of chimeric streptavidin for protein-enabled dual gold catalysis

Abstract

Artificial metalloenzymes (ArMs) result from anchoring an organometallic catalyst within an evolvable protein scaffold. Thanks to its dimer of dimers quaternary structure, streptavidin allows the precise positioning of two metal cofactors to activate a single substrate, thus expanding the reaction scope accessible to ArMs. To validate this concept, we report herein on our efforts to engineer and evolve an artificial hydroaminase based on dual-gold activation of alkynes. Guided by modelling, we designed a chimeric streptavidin equipped with a hydrophobic lid shielding its active site which enforces the advantageous positioning of two synergistic biotinylated gold cofactors. Three rounds of directed evolution using E. coli cellfree extracts led to the identification of mutants favouring either the anti-Markovnikov product (an indole carboxamide with 96% regioselectivity, 51 TONs) resulting from a dual gold activation of an ethynylphenylurea substrate or the Markovnikov product (a phenyldihydroquinazolinone with 99% regioselectivity, 333 TONs) resulting from the -activation of the alkyne by gold. genetic means. Thus far, more than 40 reactions can be catalysed by ArMs. 25 Current challenges in the field include; protein-accelerated catalysis, whereby a pre-catalyst is activated upon incorporation within the host protein, 26 dual catalysis 27,28 and compatibility of the ArM with a cytosolic environment. 29 Privileged scaffolds for ArMs include: carbonic anhydrase, 30 hemoproteins, 31,32 prolyl oligopeptidase, 33 lactococcal multiresistance regulator, 23 four helix-bundles, 34,35 nitrobindin, 36 human serum albumin, 37 and (strept)avidin. 20,[38][39][40] The work presented herein capitalizes on the unique topology of Sav enabling the localization of two close-lying biotinylated probes within a hydrophobic environment. This enabled the engineering and evolution of a biocompatible artificial hydroaminase (HAMase hereafter) based on either single-or dual-gold activation of an alkyne, Figure 1. Results Design of the artificial hydroaminaseAs reported by Asensio 5,41 and van der Vlugt 42 , the regioselectivity for the hydroamination of ethynylurea 1 is by-and-large governed by the mode of activation of the alkyne by gold: the canonical -activation favours the quinazolinone 3 (Markovnikov, 6-exo-dig addition product), while the dual -gold activation affords preferentially the indole 2 (anti-Markovnikov, 5endo-dig addition product) 5,42,43 Upon -coordination of the alkyne to gold, the pKa of the terminal C-H bond is lowered, thus favouring its deprotonation and coordination by a second gold to afford the -activation mode. 41 Accordingly, the spatial arrangement of the two gold species is critical in determining the regioselectivity of the reaction. We thus selected the gold-catalyzed cyclization of the ethynylurea 1 to engineer and evolve a dual-gold catalysed hydroaminase (HAMase) based on the biotin-streptavidin technology.Thanks to its dimer of dimers quaternary structure, which places the valeric acid side ch...

Controlling the optical and catalytic properties of artificial metalloenzyme photocatalysts using chemogenetic engineering

“…More open scaffolds like streptavidin do not fully encapsulate anchored catalysts, 64 so their ability to modulate cofactor properties and control reactivity is limited (though fused lid structures such as that recently reported for streptavidin could correct this issue). 65 We previously established that Pfu POP is a versatile scaffold for ArM formation 66 and that directed evolution can be used to engineer selective ArMs for a variety of dirhodium-catalyzed carbene addition and insertion reactions. 38,40 We have also used this scaffold to develop an artificial enzyme containing an acridinium cofactor ( Fig.…”

Transition Metal Scaffolds Used To Bring New‐to‐Nature Reactions into Biological Systems

“…In terms of protein‐based scaffolds, it would be nearly impossible to overlook the influence that streptavidin/avidin has had since the Whiteside group first introduced an avidin ArM in 1978 [148] . The main advantage of avidin/streptavidin are their strong affinities to the small molecule biotin, which can be used to easily anchor different metal complexes to catalyze various transformations [47,148–191] . While these reactions and subsequent applications are best explained in other review articles, [192,193] it is worth noting a recent example involving the development of DNA protocells (PC) [176] .…”

A Plurizyme with Transaminase and Hydrolase Activity Catalyzes Cascade Reactions

“…They have provided new solutions for activating and transforming specific molecules involving several steps, or even new-to-nature reactions. [14][15][16] The challenge originates from the difficulty in precisely controlling the positioning of the catalytic entities in a single protein scaffold, which is different from combining the active centers of two covalently bound enzymes. [11][12][13] The catalytic potential of an artificial protein scaffold with two abiotic catalytic groups has been recently demonstrated by the design of a chimeric streptavidin with two adjacent Au I complexes structurally equal.…”

“…The catalytic potential of an artificial protein scaffold with two abiotic catalytic groups has been recently demonstrated by the design of a chimeric streptavidin with two adjacent Au I complexes structurally equal. Both gold entities can work individually but, through adopting multiple poses, can work in synergy to activate an alkyne [14, 17] . In a recent elegant study, the incorporation of a serine close to the haem cofactor of a P450 enzyme allowed the design of a dual‐function catalyst for efficient enantioselective carbene C−H insertion reactions [15] .…”