title: Fusion of a flavin-based fluorescent protein to hydroxynitrile lyase from Arabidopsis thaliana improves enzyme stability.
abstract: Hydroxynitrile 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.
authors.affiliation: Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf and Forschungszentrum Jülich, Jülich, Germany.
authors.forename: Kathrin Emmi
authors.initials: KE
authors.lastname: Scholz
authors.forename: Benita
authors.initials: B
authors.lastname: Kopka
authors.forename: Astrid
authors.initials: A
authors.lastname: Wirtz
authors.forename: Martina
authors.initials: M
authors.lastname: Pohl
authors.forename: Karl-Erich
authors.initials: KE
authors.lastname: Jaeger
authors.forename: Ulrich
authors.initials: U
authors.lastname: Krauss
mesh_terms: D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005415:Flavins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D005415:Flavins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000446:Aldehyde-Lyases; C021541:mandelonitrile lyase