ChemInform Abstract: An R‐Selective Hydroxynitrile Lyase from Arabidopsis thaliana with an α/β‐Hydrolase Fold.

Andexer, Jennifer N.; Langermann, Jan von; Mell, Annett; Bocola, Marco; Kragl, Udo; Eggert, Thorsten; Pohl, Martina

doi:10.1002/chin.200812033

Cited by 13 publications

(32 citation statements)

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“…With increasing reaction times, conversion and ee values dropped to about 61% and 97% after 60 min, respectively. This effect is most probably related to nonenzymatic cleavage of the produced (R)-chloromandelonitrile, which is more pronounced at the employed buffer pH than that of (R)-mandelonitrile (26). Initial reaction rates and obtained ee values are thus superior to conversion of the same substrate by our previously described whole-cell biotransformation approach, where maximally 80% 2-chlorobenzaldehyde was converted within 60 min of reaction time, with a moderate ee of 70% (wet E. coli cells expressing AtHNL wild type) in monophasic MTBE as a solvent system.…”

Section: Discussionmentioning

confidence: 96%

“…Under those conditions, complete conversion of benzaldehyde and HCN to (R)-mandelonitrile was observed within 120 min, with the nonenzymatic side reaction amounting to about 14% (26). Hence, our stabilized FbFP-AtHNL fusions might represent an interesting alternative to wild-type AtHNL, i.e., for the conversion of more demanding substrates, such as 2-chlorobenzaldehyde and 2-bromobenzaldehyde, that show faster nonenzymatic reaction rates than benzaldehyde (26). To address this issue, we followed the conversion of 2-chlorobenzaldehyde using cFbFP-AtHNL as a catalyst.…”

Section: Discussionmentioning

confidence: 98%

“…The AtHNL-catalyzed synthesis of chiral cyanohydrins has been carried out in the past using various purified and immobilized enzyme preparations (15,17,18,26). Hereby, the enzyme has mostly been used either in micro-aqueous reaction systems as an immobilized or whole-cell biocatalyst (15,18) or in purified form in an aqueous-organic two-phase system (26).…”

Section: Discussionmentioning

confidence: 99%

“…Hereby, the enzyme has mostly been used either in micro-aqueous reaction systems as an immobilized or whole-cell biocatalyst (15,18) or in purified form in an aqueous-organic two-phase system (26). In the latter system, the buffer pH was set to 5 and diisopropyl ether was used as the organic phase (ratio, 1:1) (26).…”

Section: Discussionmentioning

confidence: 99%

“…The stereoselectivity of HNL-catalyzed hydrocyanation reactions is strongly influenced by the employed reaction conditions (see Fig. S1 in the supplemental material) (26). In aqueous media, the limiting factor, compromising the optical purity of the enzymatic cyanohydrin formation, is a nonenzymatic hydrocyanation reaction which results in the for- (B) Influence of pH on the relative initial rate activity.…”

Section: Construction and Heterologous Overexpression Of Fluorescent mentioning

confidence: 99%

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Fusion of a Flavin-Based Fluorescent Protein to Hydroxynitrile Lyase from Arabidopsis thaliana Improves Enzyme Stability

Scholz

Kopka

Wirtz

et al. 2013

Appl Environ Microbiol

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bHydroxynitrile lyase from Arabidopsis thaliana (AtHNL) was fused to different fluorescent reporter proteins. Whereas all fusion constructs retained enzymatic activity and fluorescence in vivo and in vitro, significant differences in activity and pH stability were observed. In particular, flavin-based fluorescent reporter (FbFP) fusions showed almost 2 orders of magnitude-increased half-lives in the weakly acidic pH range compared to findings for the wild-type enzyme. Analysis of the quaternary structure of the respective FbFP-AtHNL fusion proteins suggested that this increased stability is apparently caused by oligomerization mediated via the FbFP tag. Moreover, the increased stability of the fusion proteins enabled the efficient synthesis of (R)-mandelonitrile in an aqueous-organic two-phase system at a pH of <5. Remarkably, (R)-mandelonitrile synthesis is not possible using wild-type AtHNL under the same conditions due to the inherent instability of this enzyme below pH 5. The fusion strategy presented here reveals a surprising means for the stabilization of enzymes and stresses the importance of a thorough in vitro characterization of in vivo-employed fluorescent fusion proteins. For scientific reasons and biotechnological purposes, it is of prime importance to label certain proteins within a cell in order to track their fate without impairing their function. To this end, fluorescent reporters can be translationally fused to the target protein (1). Ideally, the labeled target protein retains its function and at the same time becomes spectroscopically and microscopically traceable within the cell (1, 2).The green fluorescent protein (GFP) and its derivatives have in the past been widely used as translationally fused reporter proteins for the analysis of expression, localization, folding, movement, and interaction of proteins in a wide variety of cells and tissues (for a recent review, see reference 2).Flavin-based fluorescent proteins (FbFPs), derived from plant and bacterial light, oxygen, voltage (LOV) photoreceptors (3), represent a promising new class of fluorescent proteins with great potential for biotechnological and biomedical applications (4). In contrast to the case for the GFP family of proteins, FbFP fluorescence does not depend on the presence of molecular oxygen (5). Therefore, bacterial FbFPs have been used as reporter proteins under hypoxic and anoxic conditions in prokaryotic (5-7) and eukaryotic (8) unicellular organisms and in mammalian cells (9). Plant-derived FbFPs, such as the iLOV (LOV domain with improved fluorescent properties) protein and its derivatives (10, 11), have previously been used as reporters of plant virus infection (10). Furthermore, the monomeric iLOV protein was recently applied as a C-terminal tag fused translationally to several target proteins (12). The majority of the tested iLOV fusion proteins could be expressed in fluorescent form, and target protein functionality was proven in one case (12). To the best of our knowledge, bacterial FbFPs have so far not been used...

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Construction and Heterologous Overexpression Of Fluorescent mentioning

confidence: 99%

See 3 more Smart Citations

Fusion of a Flavin-Based Fluorescent Protein to Hydroxynitrile Lyase from Arabidopsis thaliana Improves Enzyme Stability

Scholz

Kopka

Wirtz

et al. 2013

Appl Environ Microbiol

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A QM/MM study of the reaction mechanism of (R)‐hydroxynitrile lyases from Arabidopsis thaliana (AtHNL)

2014

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Hydroxynitrile lyases (HNLs) catalyze the conversion of chiral cyanohydrins to hydrocyanic acid (HCN) and aldehyde or ketone. Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) is the first R-selective HNL enzyme containing an α/β-hydrolases fold. In this article, the catalytic mechanism of AtHNL was theoretically studied by using QM/MM approach based on the recently obtained crystal structure in 2012. Two computational models were constructed, and two possible reaction pathways were considered. In Path A, the calculation results indicate that the proton transfer from the hydroxyl group of cyanohydrin occurs firstly, and then the cleavage of C1-C2 bond and the rotation of the generated cyanide ion (CN(-)) follow, afterwards, CN(-) abstracts a proton from His236 via Ser81. The C1-C2 bond cleavage and the protonation of CN(-) correspond to comparable free energy barriers (12.1 vs. 12.2 kcal mol(-1)), suggesting that both of the two processes contribute a lot to rate-limiting. In Path B, the deprotonation of the hydroxyl group of cyanohydrin and the cleavage of C1-C2 bond take place in a concerted manner, which corresponds to the highest free energy barrier of 13.2 kcal mol(-1). The free energy barriers of Path A and B are very similar and basically agree well with the experimental value of HbHNL, a similar enzyme of AtHNL. Therefore, both of the two pathways are possible. In the reaction, the catalytic triad (His236, Ser81, and Asp208) acts as the general acid/base, and the generated CN(-) is stabilized by the hydroxyl group of Ser81 and the main-chain NH-groups of Ala13 and Phe82.

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Hydroxynitrile lyase catalyzed cyanohydrin synthesis at high pH-values

Langermann

Guterl

Pohl

et al. 2008

Bioprocess Biosyst Eng

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The application of unusual high pH-values within enzymatic cyanohydrin synthesis has been investigated. Usually enzymatic cyanohydrin synthesis in two-phase systems requires low pH-values within the aqueous phase to suppress the non-enzymatic side reaction. In contrast, we investigated the usage of pH-values above pH 6 by using the highly enantioselective (S)-selective hydroxynitrile lyase from Manihot esculenta. With these unusual reaction conditions also the unfavorable substrate 3-phenoxy-benzaldehyde can be converted by the wild type enzyme with excellent conversion and enantiomeric excess yielding pure (S)-3-phenoxy-benzaldehyde cyanohydrin with an enantiomeric excess of 97%. Although the variant MeHNL-W128A shows a higher activity with respect to this reaction, the enantioselectivity was reduced (85% e.e.(S)). Additionally, a new continuous spectroscopic cyanohydrin assay monitoring the formation of 3-phenoxy-benzaldehyde cyanohydrin was developed.

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ChemInform Abstract: An R‐Selective Hydroxynitrile Lyase from Arabidopsis thaliana with an α/β‐Hydrolase Fold.

Cited by 13 publications

References 1 publication

Fusion of a Flavin-Based Fluorescent Protein to Hydroxynitrile Lyase from Arabidopsis thaliana Improves Enzyme Stability

Fusion of a Flavin-Based Fluorescent Protein to Hydroxynitrile Lyase from Arabidopsis thaliana Improves Enzyme Stability

A QM/MM study of the reaction mechanism of (R)‐hydroxynitrile lyases from Arabidopsis thaliana (AtHNL)

Hydroxynitrile lyase catalyzed cyanohydrin synthesis at high pH-values

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