Complete switch of reaction specificity of an aldolase by directed evolution in vitro: Synthesis of generic aliphatic aldol products
Authors: Wolf-Dieter Fessner, Sebastian Junker, Raquel Roldan, Henk-Jan Joosten, Pere Clapés
Journal: Angewandte Chemie International Edition
A structure‐guided engineering of fructose‐6‐phosphate aldolase was performed to expand its substrate promiscuity toward aliphatic nucleophiles, i.e., unsubstituted alkanones and alkanals. A "smart" combinatorial library was created targeting residues D6, T26 and N28 that form a binding pocket around the nucleophilic carbon atom. Double‐selectivity screening was executed by high‐performance TLC that allowed simultaneous determination of total activity as well as a preference for acetone versus propanal as competing nucleophiles. While any mutation of N28 resulted in inactivation of the enzyme, D6 turned out to be the key residue that enabled activity with non‐hydroxylated nucleophiles. Altogether 25 single‐ and double‐site variants (D6X and D6X/T26X) were discovered that show useful synthetic activity and a varying preference for ketone or aldehyde as the aldol nucleophiles. Remarkably, all of the novel variants had completely lost their native activity for cleavage of fructose 6‐phosphate.
Mathematical model of the MenD-catalyzed 1,4-addition (Stetter reaction) of α-ketoglutaric acid to acrylonitrile
Authors: Martina Sudar, Đurđa Vasić-Rački, Michael Müller, Alexandra Walter, Zvjezdana Findrik Blažević
Journal: Journal of Biotechnology
The Stetter reaction, a conjugate umpolung reaction, is well known for cyanide-catalyzed transformations of mostly aromatic aldehydes. Enzymatic Stetter reactions, however, have been largely unexplored, especially with respect to preparative transformations. We have investigated the kinetics of the MenD-catalyzed 1,4-addition of α-ketoglutaric acid to acrylonitrile which has shown that acrylonitrile, while an interesting candidate, is a poor substrate for MenD due to low affinity of the enzyme for this substrate. The kinetic model of the reaction was simplified to double substrate Michaelis–Menten kinetics where the reaction rate linearly depends on acrylonitrile concentration. Experiments at different initial concentrations of acrylonitrile under batch, repetitive batch, and fed-batch reactor conditions were carried out to validate the developed mathematical model. Thiamine diphosphate dependent MenD proved to be quite a robust enzyme; nevertheless, enzyme operational stability decay occurs in the reactor. The spontaneous reactivity of acrylonitrile towards polymerization was also taken into account during mathematical modeling. Almost quantitative conversion of acrylonitrile was achieved in all batch reactor experiments, while the yield of the desired product was dependent on initial acrylonitrile concentration (i.e., the concentration of the stabilizer additive). Using the optimized reactor parameters, it was possible to synthesize the product, 6-cyano-4-oxohexanoic acid, in a concentration of 250 mM. The highest concentration of product was achieved in a repetitive batch reactor experiment. A fed-batch reactor experiment also delivered promising results, especially regarding the short reaction time needed to achieve a 200 mM concentration of product. Hence, the enzymatic Stetter reaction with a highly reactive acceptor substrate can be performed on a preparative scale, which should enable similar transformations with acrylate, methacrylate, and methyl vinyl ketone.
Authors: Pere Clapes, Jesús Joglar, Jordi Bujons, Teodor Parella, Karel Hernánde
Journal: Angewandte Chemie International Edition
Year of publication: 2018
Pyruvate-dependent aldolases exhibit a stringent selectivity for pyruvate, limiting their synthetic potential application, a drawback shared with other existing aldolases. Structure-guided rational protein engineering rendered a 2-keto-3-deoxy-L-rhamnonate aldolase variant, fused with maltose binding protein (MBP-YfaU W23V/L216A), capable to efficiently convert larger pyruvate analogs, e.g. having linear and branched aliphatic chains, in aldol addition reactions. Combination of these nucleophiles with N-Cbz-alaninal and N-Cbz-prolinal electrophiles gave access to chiral building blocks, e.g. derivatives of (2S,3S,4R)-4-amino-3-hydroxy-2-methylpentanoic acid (68%, dr 90:10) and the enantiomer of Dolaproine (33%, dr 94:6) as well as a collection of unprecedented α-amino acid derivatives of the proline and pyrrolizidine type, with conversions varying between 6-93% and diasteromeric ratios from 50:50 to 95:5 depending on the nucleophilic and electrophilic components
2-Keto-3-Deoxy-l-Rhamnonate Aldolase (YfaU) as Catalyst in Aldol Additions of Pyruvate to Amino Aldehyde Derivatives
4-Hydroxy-2-keto acid derivatives are versatile building blocks for the synthesis of amino acids, hydroxy carboxylic acids and chiral aldehydes. Pyruvate aldolases are privileged catalysts for a straightforward access to this class of keto acid compounds. In this work, a Class II pyruvate aldolase from Escherichia coli K-12, 2-keto-3-deoxy-l-rhamnonate aldolase (YfaU), was evaluated for the synthesis of amino acid derivatives of proline, pipecolic acid, and pyrrolizidine-3-carboxylic acid. The aldol addition of pyruvate to N-protected amino aldehydes was the key enzymatic aldol addition step followed by catalytic intramolecular reductive amination. The corresponding N-Cbz-amino-4-hydroxy-2-keto acid (Cbz=benzyloxycarbonyl) precursors were obtained in 51–95% isolated yields and enantioselectivity ratios from 26:74 to 95:5, with chiral α-substituted N-Cbz-amino aldehydes. (S)-N-Cbz-amino aldehydes gave aldol adducts with preferentially (R)-configuration at the newly formed stereocenter, whereas the contrary is true for (R)-N-Cbz-amino aldehydes. Addition reactions to achiral amino aldehydes rendered racemic aldol adducts. Molecular models of the pre-reaction ternary complexes YfaU-pyruvate enolate-acceptor aldehyde were constructed to explain the observed stereochemical outcome of the reactions. Catalytic reductive amination of the aldol adducts yielded 4-hydroxy-2-pipecolic acid, and unprecedented C-5 substituted 4-hydroxyproline and pyrrolizidine-3-carboxylic acid derivatives.
Fluorogenic Kinetic Assay for High-Throughput Discovery of Stereoselective Ketoreductases Relevant to Pharmaceutical Synthesis
Authors: Yen-Chi Thai, Anna Szekrenyi, Yuyin Qi, Gary W. Black, Simon J. Charnock, Wolf- Dieter Fessner
Journal: Bioorganic & Medicinal Chemistry
Year of publication: 2017
Enantiomerically pure 1-(6-methoxynaphth-2-yl) and 1-(6-(dimethylamino)naphth-2-yl) carbinols are fluorogenic substrates for aldo/keto reductase (KRED) enzymes, which allow the highly sensitive and reliable determination of activity and kinetic constants of known and unknown enzymes, as well as an immediate enantioselectivity typing. Because of its simplicity in microtiter plate format, the assay qualifies for the discovery of novel KREDs of yet unknown specificity among this vast enzyme superfamily. The suitability of this approach for enzyme typing is illustrated by an exemplary screening of a large collection of short-chain dehydrogenase/reductase (SDR) enzymes arrayed from a metagenomic approach. We believe that this assay format should match well the pharmaceutical industry’s demand for acetophenone-type substrates and the continuing interest in new enzymes with broad substrate promiscuity for the synthesis of chiral, non-racemic carbinols.
Amino acids are of paramount importance as chiral building blocks of life, for drug development in modern medicinal chemistry, and for the manufacture of industrial products. In this work, the stereoselective synthesis of (S)- and (R)-2-amino-4-hydroxybutanoic acid was accomplished using a Systems Biocatalysis approach comprising a biocatalytic one-pot cyclic cascade by coupling of an aldol reaction with an ensuing stereoselective transamination. A Class II pyruvate aldolase from E. coli, expressed as a soluble fusion protein, in tandem with either an (S)- or (R)-selective, pyridoxal phosphate-dependent, transaminase were used as catalysts to realize the conversion, with formaldehyde and alanine being the sole starting materials. Interestingly, the Class II pyruvate aldolase was found to tolerate for-maldehyde concentrations of up to 1.4 M. The cascade system was found to reach product concentrations for (S)- or (R)-2-amino-4-hydroxybutanoic acid of at least 0.4 M, rendering yields between 86% and >95%, respectively, productivities of >80 g L–1 d–1, and ee >99%
Droplet microfluidics will become a disruptive technology in the field of library screening and replace biological selections if the central dogma of biology and other processes are successfully implemented within microdroplets.
The increasing number of enzyme applications in chemical synthesis calls for new engineering methods to develop the biocatalysts of the future. An interesting concept in enzyme engineering is the generation of large-scale mutational data in order to chart protein mutability landscapes. These landscapes allow the important discrimination between beneficial mutations and those that are neutral or detrimental, thus providing detailed insight into sequence–function relationships. As such, mutability landscapes are a powerful tool with which to identify functional hotspots at any place in the amino acid sequence of an enzyme. These hotspots can be used as targets for combinatorial mutagenesis to yield superior enzymes with improved catalytic properties, stability, or even new enzymatic activities. The generation of mutability landscapes for multiple properties of one enzyme provides the exciting opportunity to select mutations that are beneficial either for one or for several of these properties. This review presents an overview of the recent advances in the construction of mutability landscapes and discusses their importance for enzyme engineering.
Recent advances on halohydrin dehalogenases—from enzyme identification to novel biocatalytic applications
Halohydrin dehalogenases are industrially relevant enzymes that catalyze the reversible dehalogenation of vicinal haloalcohols with formation of the corresponding epoxides. In the reverse reaction, also other negatively charged nucleophiles such as azide, cyanide, or nitrite are accepted besides halides to open the epoxide ring. Thus, novel C-N, C-C, or C-O bonds can be formed by halohydrin dehalogenases, which makes them attractive biocatalysts for the production of various β-substituted alcohols. Despite the fact that only five individual halohydrin dehalogenase enzyme sequences have been known until recently enabling their heterologous production, a large number of different biocatalytic applications have been reported using these enzymes. The recent characterization of specific sequence motifs has facilitated the identification of novel halohydrin dehalogenase sequences available in public databases and has largely increased the number of recombinantly available enzymes. These will help to extend the biocatalytic repertoire of this enzyme family and to foster novel biotechnological applications and developments in the future. This review gives a general overview on the halohydrin dehalogenase enzyme family and their biochemical properties and further focuses on recent developments in halohydrin dehalogenase biocatalysis and protein engineering.