Uneven twins: Comparison of two enantiocomplementary hydroxynitrile lyases with α/β-hydrolase fold

Guterl, Jan‐Karl; Andexer, Jennifer N.; Sehl, Torsten; Langermann, Jan von; Frindi-Wosch, Ilona; Rosenkranz, Tobias; Fitter, Jörg; Gruber, Karl; Kragl, Udo; Eggert, Thorsten; Pohl, Martina

doi:10.1016/j.jbiotec.2009.03.010

Cited by 55 publications

(70 citation statements)

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“…Since SacI was used to clone the respective FbFP fragment into the pET28a vector, the AtHNL-encoding gene fragment could be inserted into the FbFP-carrying pETnFbFP and pETcFbFP plasmid constructs. The HNLencoding gene fragment was PCR amplified from a previously described expression plasmid (19). To generate the cFbFP-AtHNL-encoding gene fusion, the oligonucleotides cHNL_NheI_fw and cHNL_blunt_rev were used.…”

Section: Methodsmentioning

confidence: 99%

“…After 3 h of incubation at 37°C under constant agitation (120 rpm), the culture was further incubated at 15°C for an additional 72 h. Subsequently, cells were harvested at 6,750 ϫ g (30 min, 4°C). Wild-type AtHNL was expressed and purified as described previously (19).…”

Section: Methodsmentioning

confidence: 99%

“…For determination of the native molecular weights (MW), a mixture of standard proteins (each 1 mg/ml) (aqueous SEC1 Photometric mandelonitrile cleavage assay for quantification of HNL activity. Mandelonitrile cleavage reactions were carried out as described previously (19). In brief, the accumulation of benzaldehyde, which is formed as one reaction product of the HNL-catalyzed cleavage of rac-mandelonitrile, was photometrically monitored at 280 nm.…”

Section: Methodsmentioning

confidence: 99%

“…The reaction mixture was constantly mixed using a magnetic stirrer. At defined time intervals, samples were withdrawn and (R)-mandelonitrile or (R)-chloromandelonitrile formation was monitored by chiral GC as described previously (18,19).…”

Section: Methodsmentioning

confidence: 99%

“…mation of racemic product (see Fig. S1) (19). This nonenzymatic reaction can be suppressed by using low pH values, by lowering the reaction temperature, and by decreasing the water content in the reaction system (18).…”

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: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Construction and Heterologous Overexpression Of Fluorescent mentioning

confidence: 99%

See 3 more Smart Citations