Enantioselective Enzyme-Catalysed Synthesis of Cyanohydrins

Holt, Jarle; Hanefeld, Ulf

doi:10.2174/157017909787314858

Cited by 121 publications

(58 citation statements)

References 2 publications

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“…As it is known that a chemical background reaction proceeds readily in aqueous medium [5], a buffer control, as well as cleared lysate of E. coli without GtHNL (empty vector control), were used as controls. The lysate of the empty vector control showed 15% conversion after 6 h, compared with 80% for the lysate expressing GtHNL, and the control reaction showed no enantioselectivity.…”

Section: Enantioselective Synthesis Of Mandelonitrilementioning

confidence: 99%

“…The protein crystals diffracted to~2. 5 A, and contained two homotetramers in the asymmetric unit (Fig. 5A).…”

Section: Overall Three-dimensional Structure Of Gthnlmentioning

confidence: 99%

“…In chemical synthesis, reversal of the natural cleavage reaction is employed. Cyanohydrins constitute important building blocks for fine chemicals, as their synthesis creates a new stereocentre and adds a versatile functional group (the nitrile group) at the same time [4][5][6]. HNLs comprise a diverse group of enzymes that vary in terms of their substrate specificity, enantioselectivity and the need for a co-factor.…”

Section: Introductionmentioning

confidence: 99%

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Biochemical and structural characterization of a novel bacterial manganese‐dependent hydroxynitrile lyase

et al. 2013

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Hydroxynitrile lyases (HNLs), which catalyse the decomposition of cyanohydrins, are found mainly in plants. In vitro, they are able to catalyse the synthesis of enantiopure cyanohydrins, which are versatile building blocks in the chemical industry. Recently, HNLs have also been discovered in bacteria. Here, we report on the detailed biochemical and structural characterization of a hydroxynitrile lyase from Granulicella tundricola (GtHNL), which was successfully heterologously expressed in Escherichia coli. The crystal structure was solved at a crystallographic resolution of 2.5 Å and exhibits a cupin fold. As GtHNL does not show any sequence or structural similarity to any other HNL and does not contain conserved motifs typical of HNLs, cupins represent a new class of HNLs. GtHNL is metal‐dependent, as confirmed by inductively coupled plasma/optical emission spectroscopy, and in the crystal structure, manganese is bound to three histidine and one glutamine residue. GtHNL displayed a specific activity of 1.74 U·mg−1 at pH 6 with (R)‐mandelonitrile, and synthesized (R)‐mandelonitrile with 90% enantiomeric excess at 80% conversion using 0.5 m benzaldehyde in a biphasic reaction system with methyl tertiary butyl ether.

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Section: Enantioselective Synthesis Of Mandelonitrilementioning

confidence: 99%

“…The protein crystals diffracted to~2. 5 A, and contained two homotetramers in the asymmetric unit (Fig. 5A).…”

Section: Overall Three-dimensional Structure Of Gthnlmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Biochemical and structural characterization of a novel bacterial manganese‐dependent hydroxynitrile lyase

et al. 2013

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“…They are of great relevance in organic synthesis, in which the ability of the enzymes to catalyze the reverse reaction forming C-C bonds in a stereoselective manner is of substantial industrial relevance (9,11,19). Hydroxynitrile lyases are quite heterogeneous exhibiting remarkable diversity with respect to their substrate specificity, mass, glycosylation, and amino acid sequence, and their similarity to oxidoreductases, ␣/␤-hydrolases, carboxypeptidases, or Zn 2ϩ -containing alcohol dehydrogenases has been reported (3,4,8,17,18,19).…”

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confidence: 99%

Characterization of Two Bacterial Hydroxynitrile Lyases with High Similarity to Cupin Superfamily Proteins

Hussain

Wiedner

Steiner

et al. 2012

Appl Environ Microbiol

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Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins. In the reverse reaction, they catalyze the formation of carbon-carbon bonds by enantioselective condensation of hydrocyanic acid with carbonyls. In this study, we describe two proteins from endophytic bacteria that display activity in the cleavage and the synthesis reaction of (R)-mandelonitrile with up to 74% conversion of benzaldehyde (enantiopreference ee 89%). Both showed high similarity to proteins of the cupin superfamily which so far were not known to exhibit HNL activity. Hydroxynitrile lyases (HNLs) catalyze the reversible cleavage of cyanohydrins, yielding the respective carbonyl compound and HCN. They are of great relevance in organic synthesis, in which the ability of the enzymes to catalyze the reverse reaction forming C-C bonds in a stereoselective manner is of substantial industrial relevance (9,11,19). Hydroxynitrile lyases are quite heterogeneous exhibiting remarkable diversity with respect to their substrate specificity, mass, glycosylation, and amino acid sequence, and their similarity to oxidoreductases, ␣/␤-hydrolases, carboxypeptidases, or Zn 2ϩ -containing alcohol dehydrogenases has been reported (3,4,8,17,18,19).Hydroxynitrile lyases are widespread in plants playing a major role in disease suppression, and only recently a bacterial protein with HNL activity in the cyanohydrin cleavage reaction was reported (16). Usually plants are colonized by a range of different bacteria (14). Endophytes in particular live in close association with their host and promote plant growth and health by various mechanisms, including the production of substances with phytohormonal activity or antimicrobial substances such as antibiotics (13,15,21). The production of HCN in pseudomonads has been reported (5), for example, but it has also been reported in other bacteria. In contrast to plants, cyanogenesis in bacteria usually follows a completely different biosynthetic pathway involving a HCN synthase (2,20). In this work, we present the discovery of a new bacterial enzyme class with hydroxynitrile lyase activity initially identified by function-based screening of a gene library of an isolated bacterial endophyte related to Pseudomonas mephitica and further investigated with a second highly similar protein from Burkholderia phytofirmans strain PsJN.Gene libraries of different endophytic bacteria isolated from potato were constructed in the pZero-2 vector by standard procedures (see supplemental material) and screened for HNL activity toward (R/S)-mandelonitrile using a colony-based colorimetric filter assay [67 mM (R/S)-mandelonitrile in 30 mM citratephosphate buffer (pH 3.5) at room temperature (RT)] (10). In the gene library of a strain related to Pseudomonas mephitica, HNL activity was detected. After subcloning and rescreening, the transformant exhibiting the strongest activity toward mandelonitrile was selected for further characterization. Sequencing revealed that the plasmid contained a 1,659-bp insert, which carried two open reading fram...

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“…A "bienzymatic approach" is considered to be especially useful for the conversion of aromatic ketones, because oxynitrilases often do not quantitatively convert these substrates, as the reaction equilibrium is largely on the side of the ketones (5,7,15,49). Furthermore, it was recently stated that the great challenge in the area of enantioselective enzyme-catalyzed cyanohydrin synthesis is the same challenge that the transition metal catalysts face: the efficient, high-yielding, and highly enantioselective conversion of ketones (17). Thus, it is reasonable to assume that the presence of a second (enantioselective) enzyme, such as a nitrilase, might result in the establishment of an efficient cascade reaction, which could circumvent the incomplete conversion of (aromatic) ketones by shifting the equilibrium to the product side.…”

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confidence: 99%

Conversion of Sterically Demanding α,α-Disubstituted Phenylacetonitriles by the Arylacetonitrilase from Pseudomonas fluorescens EBC191

Baum

Williamson

Sewell

2012

Appl Environ Microbiol

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The nitrilase from Pseudomonas fluorescens EBC191 converted 2-methyl-2-phenylpropionitrile, which contains a quaternary carbon atom in the ␣-position toward the nitrile group, and also similar sterically demanding substrates, such as 2-hydroxy-2-phenylpropionitrile (acetophenone cyanohydrin) or 2-acetyloxy-2-methylphenylacetonitrile. 2-Methyl-2-phenylpropionitrile was hydrolyzed to almost stoichiometric amounts of the corresponding acid. Acetophenone cyanohydrin was transformed to the corresponding acid (atrolactate) and amide (atrolactamide) at a ratio of about 3.4:1. The (R)-acid and the (S)-amide were formed preferentially from acetophenone cyanohydrin. A homology model of the nitrilase suggested that steric hindrance with amino acid residue Tyr54 could impair the binding or conversion of sterically demanding substrates. Therefore, several enzyme variants that carried mutations in the respective residues were generated and subsequently analyzed for the substrate specificity and enantioselectivity of the reactions. Enzyme variants that demonstrated increased relative activities for the conversion of acetophenone cyanohydrin were identified. The chiral analysis of these reactions demonstrated peculiar reaction kinetics, which suggested that the enzyme variants converted the nonpreferred (S)-enantiomer of acetophenone cyanohydrin with a higher reaction rate than that of the (preferred) (R)-enantiomer. Recombinant whole-cell catalysts that simultaneously produced the nitrilase from P. fluorescens EBC191 and a plant-derived (S)-oxynitrilase from cassava (Manihot esculenta) converted acetophenone plus cyanide at pH 4.5 to (S)-atrolactate and (S)-atrolactamide. These recombinant cells are promising catalysts for the synthesis of stable chiral quaternary carbon centers from ketones.

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Enantioselective Enzyme-Catalysed Synthesis of Cyanohydrins

Cited by 121 publications

References 2 publications

Biochemical and structural characterization of a novel bacterial manganese‐dependent hydroxynitrile lyase

Biochemical and structural characterization of a novel bacterial manganese‐dependent hydroxynitrile lyase

Characterization of Two Bacterial Hydroxynitrile Lyases with High Similarity to Cupin Superfamily Proteins

Conversion of Sterically Demanding α,α-Disubstituted Phenylacetonitriles by the Arylacetonitrilase from Pseudomonas fluorescens EBC191

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