Engineered Artificial Carboligases Facilitate Regioselective Preparation of Enantioenriched Aldol Adducts.

Controlling regio- and stereoselectivity of aldol additions is generally challenging. Here we show that an artificial aldolase with high specificity for acetone as the aldol donor can be reengineered via single active site mutations to accept linear and cyclic aliphatic ketones with notable efficiency, regioselectivity, and stereocontrol. Biochemical and crystallographic data show how the mutated residues modulate the binding and activation of specific aldol donors, as well as their subsequent reaction with diverse aldehyde acceptors. Broadening the substrate scope of this evolutionarily naïve catalyst proved much easier than previous attempts to redesign natural aldolases, suggesting that such proteins may be excellent starting points for the development of customized biocatalysts for diverse practical applications.

Stereodivergent Evolution of Artificial Enzymes for the Michael Reaction.

Enzymes are valuable biocatalysts for asymmetric synthesis due to their exacting stereocontrol. Changing the selectivity of an existing catalyst for new applications is, however, challenging. Here we show that, in contrast, the stereoselectivity of an artificial enzyme created by design and directed evolution is readily tunable. We engineered a promiscuous artificial retro-aldolase into four stereocomplementary catalysts for the Michael addition of a tertiary carbanion to an unsaturated ketone. Notably, this selectivity is also preserved with alternative Michael nucleophiles. Complete stereodiversification of other designer enzymes should similarly be possible by extension of these approaches.

Enantiocomplementary Synthesis of γ-Nitroketones Using Designed and Evolved Carboligases.

Artificial enzymes created by computational design and directed evolution are versatile biocatalysts whose promiscuous activities represent potentially attractive starting points for divergent evolution in the laboratory. The artificial aldolase RA95.5-8, for example, exploits amine catalysis to promote mechanistically diverse carboligations. Here we report that RA95.5-8 variants catalyze the asymmetric synthesis of γ-nitroketones via two alternative enantiocomplementary Michael-type reactions: enamine-mediated addition of acetone to nitrostyrenes, and nitroalkane addition to conjugated ketones activated as iminium ions. In addition, a cascade of three aldolase-catalyzed reactions enables one-pot assembly of γ-nitroketones from three simpler building blocks. Together, our results highlight the chemical versatility of artificial aldolases for the practical synthesis of important chiral synthons.

Chemoselective Henry Condensations Catalyzed by Artificial Carboligases.

The promiscuity of de novo designed enzymes provides a privileged platform for diverse abiological reactions. In this work, we report the first example of a nitroolefin synthase that catalyzes the Henry condensation between aromatic aldehydes and nitromethane. Significant catalytic activity was discovered in the computationally designed and evolved carboligase RA95.5-8, and mutations around the active site were shown to improve the reaction rate, demonstrating the potential to optimize the enzyme by directed evolution. This novel nitroolefin synthase could participate in complex biological cascades, whereby the highly tunable chemoselectivity could afford useful synthetic building blocks.

Fast Knoevenagel Condensations Catalyzed by an Artificial Schiff-Base-Forming Enzyme.

The simple catalytic motifs utilized by enzymes created by computational design and directed evolution constitute a potentially valuable source of chemical promiscuity. Here we show that the artificial retro-aldolase RA95.5-8 is able to use a reactive lysine in a hydrophobic pocket to accelerate promiscuous Knoevenagel condensations of electron-rich aldehydes and activated methylene donors. Optimization of this activity by directed evolution afforded an efficient enzyme variant with a catalytic proficiency of 5 × 10(11) M(-1) and a >10(8)-fold catalytic advantage over simple primary and secondary amines. Divergent evolution of de novo enzymes in this way could be a promising strategy for creating tailored biocatalysts for many synthetically useful reactions.

A Promiscuous De†Novo Retro-Aldolase Catalyzes Asymmetric Michael Additions via Schiff Base Intermediates.

Recent advances in computational design have enabled the development of primitive enzymes for a range of mechanistically distinct reactions. Here we show that the rudimentary active sites of these catalysts can give rise to useful chemical promiscuity. Specifically, RA95.5-8, designed and evolved as a retro-aldolase, also promotes asymmetric Michael additions of carbanions to unsaturated ketones with high rates and selectivities. The reactions proceed by amine catalysis, as indicated by mutagenesis and X-ray data. The inherent flexibility and tunability of this catalyst should make it a versatile platform for further optimization and/or mechanistic diversification by directed evolution.

Engineering the donor selectivity of D-fructose-6-phosphate aldolase for biocatalytic asymmetric cross-aldol additions of glycolaldehyde.

D-Fructose-6-phosphate aldolase (FSA) is a unique catalyst for asymmetric cross-aldol additions of glycolaldehyde. A combination of a structure-guided approach of saturation mutagenesis, site-directed mutagenesis, and computational modeling was applied to construct a set of FSA variants that improved the catalytic efficiency towards glycolaldehyde dimerization up to 1800-fold. A combination of mutations in positions L107, A129, and A165 provided a toolbox of FSA variants that expand the synthetic possibilities towards the preparation of aldose-like carbohydrate compounds. The new FSA variants were applied as highly efficient catalysts for cross-aldol additions of glycolaldehyde to N-carbobenzyloxyaminoaldehydes to furnish between 80-98 % aldol adduct under optimized reaction conditions. Donor competition experiments showed high selectivity for glycolaldehyde relative to dihydroxyacetone or hydroxyacetone. These results demonstrate the exceptional malleability of the active site in FSA, which can be remodeled to accept a wide spectrum of donor and acceptor substrates with high efficiency and selectivity.

Chemoenzymatic synthesis, structural study and biological activity of novel indolizidine and quinolizidine iminocyclitols.

The synthesis, conformational study and inhibitory properties of diverse indolizidine and quinolizidine iminocyclitols are described. The compounds were chemo-enzymatically synthesized by two-step aldol addition and reductive amination reactions. The aldol addition of dihydroxyacetone phosphate (DHAP) to N-Cbz-piperidine carbaldehyde derivatives catalyzed by L-rhamnulose 1-phosphate aldolase from Escherichia coli provides the key intermediates. The stereochemical outcome of both aldol addition and reductive amination depended upon the structure of the starting material and intermediates. The combination of both reactions furnished five indolizidine and six quinolizidine type iminocyclitols. A structural analysis by NMR and in silico density functional theory (DFT) calculations allowed us to determine the population of stereoisomers with the trans or cis ring fusion, as a consequence of the inversion of configuration of the bridgehead nitrogen. The trans fusion was by far the most stable, but for certain stereochemical configurations of the 3-hydroxymethyl and hydroxyl substituents both trans and cis fusion stereoisomers coexisted in different proportions. Some of the polyhydroxylated indolizidines and quinolizidines were shown to be moderate to good inhibitors against α-L-rhamnosidase from Penicillium decumbens. Indolizidines were found to be moderate inhibitors of the rat intestinal sucrase and of the exoglucosidase amyloglucosidase from Aspergillus niger. In spite of their activity against α-L-rhamnosidase, all the compounds were ineffective to inhibit the growth of the Mycobacterium tuberculosis, the causative agent of tuberculosis.

Highly efficient aldol additions of DHA and DHAP to N-Cbz-amino aldehydes catalyzed by L-rhamnulose-1-phosphate and L-fuculose-1-phosphate aldolases in aqueous borate buffer.

Aldol addition reactions of dihydroxyacetone (DHA) to N-Cbz-amino aldehydes catalyzed by L-rhamnulose-1-phosphate aldolase (RhuA) in the presence of borate buffer are reported. High yields of aldol adduct (e.g. 70-90%) were achieved with excellent (>98 : 2 syn/anti) stereoselectivity for most S or R configured acceptors, which compares favorably to the reactions performed with DHAP. The stereochemical outcome was different and depended on the N-Cbz-amino aldehyde enantiomer: the S acceptors gave the syn (3R,4S) aldol adduct whereas the R ones gave the anti (3R,4R) diastereomer. Moreover, the tactical use of Cbz protecting group allows simple and efficient elimination of borate and excess of DHA by reverse phase column chromatography or even by simple extraction. This, in addition to the use of unphosphorylated donor nucleophile, makes a useful and expedient methodology for the synthesis of structurally diverse iminocyclitols. The performance of aldol additions of dihydroxyacetone phosphate (DHAP) to N-Cbz-amino aldehydes using RhuA and L-fuculose-1-phosphate aldolase (FucA) catalyst in borate buffer was also evaluated. For FucA catalysts, including FucA F131A, the initial velocity of the aldol addition reactions using DHAP were between 2 and 10 times faster and the yields between 1.5 and 4 times higher than those in triethanolamine buffer. In this case, the retroaldol velocities measured for some aldol adducts were lower than those without borate buffer indicating some trapping effect that could explain the improvement of yields.

Structure-guided minimalist redesign of the L-fuculose-1-phosphate aldolase active site: expedient synthesis of novel polyhydroxylated pyrrolizidines and their inhibitory properties against glycosidases and intestinal disaccharidases.

A minimalist active site redesign of the L-fuculose-1-phosphate aldolase from E. coli FucA was envisaged, to extend its tolerance towards bulky and conformationally restricted N-Cbz-amino aldehyde acceptor substrates (Cbz=benzyloxycarbonyl). Various mutants at the active site of the FucA wild type were obtained and screened with seven sterically demanding N-Cbz-amino aldehydes including N-Cbz-prolinal derivatives. FucA F131A showed an aldol activity of 62 µmol h(-1) mg(-1) with (R)-N-Cbz-prolinal, whereas no detectable activity was observed with the FucA wild type. For the other substrates, the F131A mutant gave aldol activities from 4 to about 25 times higher than those observed with the FucA wild type. With regard to the stereochemistry of the reactions, the (R)-amino aldehydes gave exclusively the anti configured aldol adducts whereas their S counterparts gave variable ratios of anti/syn diastereoisomers. Interestingly, the F131A mutant was highly stereoselective both with (R)- and with (S)-N-Cbz-prolinal, exclusively producing the anti and syn aldol adducts, respectively. Molecular models suggest that this improved activity towards bulky and more rigid substrates, such as N-Cbz-prolinal, could arise from a better fit of the substrate into the hydrophobic pocket created by the F131A mutation, due to an additional π-cation interaction with the residue K205' and to efficient contact between the substrate and the mechanistically important Y113' and Y209' residues. An expedient synthesis of novel polyhydroxylated pyrrolizidines related to the hyacinthacine and alexine types was accomplished through aldol additions of dihydroxyacetone phosphate (DHAP) to hydroxyprolinal derivatives with the hyperactive FucA F131A as catalyst. The iminocyclitols obtained were fully characterised and found to be moderate to weak inhibitors (relative to 1,4-dideoxy-1,4-imino-L-arabinitol (LAB) and 1,4-dideoxy-1,4-imino-D-arabinitol (DAB)) against glycosidases and rat intestinal saccharidases.

Speeding up enzyme discovery and engineering with ultrahigh-throughput methods.

Exploring the sequence space of enzyme catalysts is ultimately a numbers game. Ultrahigh-throughput screening methods for rapid analysis of millions of variants are therefore increasingly important for investigating sequence-function relationships, searching large metagenomic libraries for interesting activities, and accelerating enzyme evolution in the laboratory. Recent applications of such technologies are reviewed here, with a particular focus on the practical benefits of droplet-based microfluidics for the directed evolution of natural and artificial enzymes. Broader implementation of such rapid, cost-effective screening technologies is likely to redefine the way enzymes are studied and engineered for academic and industrial purposes.

Emergence of a catalytic tetrad during evolution of a highly active artificial aldolase.

Designing catalysts that achieve the rates and selectivities of natural enzymes is a long-standing goal in protein chemistry. Here, we show that an ultrahigh-throughput droplet-based microfluidic screening platform can be used to improve a previously optimized artificial aldolase by an additional factor of 30 to give a >109 rate enhancement that rivals the efficiency of class I aldolases. The resulting enzyme catalyses a reversible aldol reaction with high stereoselectivity and tolerates a broad range of substrates. Biochemical and structural studies show that catalysis depends on a Lys-Tyr-Asn-Tyr tetrad that emerged adjacent to a computationally designed hydrophobic pocket during directed evolution. This constellation of residues is poised to activate the substrate by Schiff base formation, promote mechanistically important proton transfers and stabilize multiple transition states along a complex reaction coordinate. The emergence of such a sophisticated catalytic centre shows that there is nothing magical about the catalytic activities or mechanisms of naturally occurring enzymes, or the evolutionary process that gave rise to them.

Asymmetric assembly of aldose carbohydrates from formaldehyde and glycolaldehyde by tandem biocatalytic aldol reactions.

The preparation of multifunctional chiral molecules can be greatly simplified by adopting a route via the sequential catalytic assembly of achiral building blocks. The catalytic aldol assembly of prebiotic compounds into stereodefined pentoses and hexoses is an as yet unmet challenge. Such a process would be of remarkable synthetic utility and highly significant with regard to the origin of life. Pursuing an expedient enzymatic approach, here we use engineered D-fructose-6-phosphate aldolase from Escherichia coli to prepare a series of three- to six-carbon aldoses by sequential one-pot additions of glycolaldehyde. Notably, the pertinent selection of the aldolase variant provides control of the sugar size. The stereochemical outcome of the addition was also altered to allow the synthesis of L-glucose and related derivatives. Such engineered biocatalysts may offer new routes for the straightforward synthesis of natural molecules and their analogues that circumvent the intricate enzymatic pathways forged by evolution.

Serine hydroxymethyl transferase from Streptococcus thermophilus and L-threonine aldolase from Escherichia coli as stereocomplementary biocatalysts for the synthesis of beta-hydroxy-alpha,omega-diamino acid derivatives.

A novel serine hydroxymethyl transferase from Streptococcus thermophilus (SHMT) and a L-threonine aldolase from Escherichia coli (LTA) were used as stereocomplementary biocatalysts for the aldol addition of glycine to N-Cbz amino aldehydes and benzyloxyacetaldehyde (Cbz=benzyloxycarbonyl). Both threonine aldolases were classified as low-specific L-allo-threonine aldolases, and by manipulating reaction parameters, such as temperature, glycine concentration, and reaction media, SHMT yielded exclusively L-erythro diastereomers in 34-60 % conversion, whereas LTA gave L-threo diastereomers in 30:70 to 16:84 diastereomeric ratios and with 40-68 % conversion to product. SHMT is among the most stereoselective L-threonine aldolases described. This is due, among other things, to its activity-temperature dependence: at 4 degrees C SHMT has high synthetic activity but negligible retroaldol activity on L-threonine. Thus, the kinetic L-erythro isomer was largely favored and the reactions were virtually irreversible, highly stereoselective, and in turn, gave excellent conversion. It was also found that treatment of the prepared N-Cbz-gamma-amino-beta-hydroxy-alpha-amino acid derivatives with potassium hydroxide (1 m) resulted in the spontaneous formation of 2-oxazolidinone derivatives of the beta-hydroxyl and gamma-amino groups in quantitative yield. This reaction might be useful for further chemical manipulations of the products.