Expression of the Zn2+-containing hydroxynitrile lyase from flax (Linum usitatissimum) in Pichia pastoris— utilization of the recombinant enzyme for enzymatic analysis and site-directed mutagenesis

Trummler, Klaus; Roos, Jürgen; Schwaneberg, Ulrich; Effenberger, Franz; Förster, Siegfried; Pfizenmaier, Klaus; Wajant, Harald

doi:10.1016/s0168-9452(98)00173-3

Cited by 41 publications

(31 citation statements)

References 18 publications

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“…in alcohol dehydrogenases [33]. Moreover, a Zn 2+ -dependent HNL from Linum usitatissimum (LuHNL) with high sequence similarity to alcohol dehydrogenases was discovered many years ago [34]. However, no exact reaction mechanism has yet been published.…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

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

et al. 2013

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“…Acetone cyanohydrin lyase (EC 4.1.2.37), the third enzyme, found in L. usitatissimum (R), 8,9) in M. exculenta (S), 4) and in H. brasiliensis (S). 5) The characteristics of PmHNL are as follows: It contains FAD as a cofactor, is inhibited by sulfhydryl reagents, and shows high sequence homology with PsHNL and PaHNL.…”

Section: )mentioning

confidence: 99%

“…(S)-HNLs are found and investigated in Ximenia americana (XaHNL), 3) Manihot esculenta (MeHNL), 4) Hevea brasiliensis (HbHNL), 5) and Sorghum bicolor (SbHNL). 6) (R)-HNLs are Phlebodium aureum (PhaHNL), 7) Linum usitatissimum (LuHNL), 8,9) Arabidopsis thaliana (AtHNL), 10) and Pr. serotina (PsHNL), [11][12][13] and the most widely investigated HNL is from Pr.…”

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confidence: 99%

Characterization of a New (R)-Hydroxynitrile Lyase from the Japanese ApricotPrunus mumeand cDNA Cloning and Secretory Expression of One of the Isozymes inPichia pastoris

Fukuta

Nanda

Kato

et al. 2011

Bioscience, Biotechnology, and Biochemistry

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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).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).…”

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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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Expression of the Zn2+-containing hydroxynitrile lyase from flax (Linum usitatissimum) in Pichia pastoris— utilization of the recombinant enzyme for enzymatic analysis and site-directed mutagenesis

Cited by 41 publications

References 18 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 a New (R)-Hydroxynitrile Lyase from the Japanese ApricotPrunus mumeand cDNA Cloning and Secretory Expression of One of the Isozymes inPichia pastoris

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

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