Electron transfer pathways in a light, oxygen, voltage (LOV) protein devoid of the photoactive cysteine.

Blue-light absorption by the flavin chromophore in light, oxygen, voltage (LOV) photoreceptors triggers photochemical reactions that lead to the formation of a flavin-cysteine adduct. While it has long been assumed that adduct formation is essential for signaling, it was recently shown that LOV photoreceptor variants devoid of the photoactive cysteine can elicit a functional response and that flavin photoreduction to the neutral semiquinone radical is sufficient for signal transduction. Currently, the mechanistic basis of the underlying electron- (eT) and proton-transfer (pT) reactions is not well understood. We here reengineered pT into the naturally not photoreducible iLOV protein, a fluorescent reporter protein derived from the Arabidopsis thaliana phototropin-2 LOV2 domain. A single amino-acid substitution (Q489D) enabled efficient photoreduction, suggesting that an eT pathway is naturally present in the protein. By using a combination of site-directed mutagenesis, steady-state UV/Vis, transient absorption and electron paramagnetic resonance spectroscopy, we investigate the underlying eT and pT reactions. Our study provides strong evidence that several Tyr and Trp residues, highly conserved in all LOV proteins, constitute the eT pathway for flavin photoreduction, suggesting that the propensity for photoreduction is evolutionary imprinted in all LOV domains, while efficient pT is needed to stabilize the neutral semiquinone radical.

Photophysics of the LOV-Based Fluorescent Protein Variant iLOV-Q489K Determined by Simulation and Experiment.

Light, oxygen, voltage (LOV) based fluorescent proteins (FPs) represent a promising alternative to fluorescent reporters of the green fluorescent protein family. For certain applications like multicolor imaging or the design of FRET-based biosensors, the generation of spectrally shifted LOV-based FPs would be required. In a recent theoretical study ( Khrenova J. Phys. Chem. B 2015 , 119 ( 16 ), pp 5176 - 5183 ), the photophysical properties of a variant of the LOV-based fluorescent protein iLOV were predicted using quantum mechanics/molecular mechanics (QM/MM) approaches. The variant contained a lysine residue at the position of a highly conserved glutamine residue (Q489K), which directly interacts with the O4 and N5 atom of the flavin mononucleotide (FMN) chromophore. On the basis of QM/MM calculations, iLOV-Q489K was suggested to possess substantially red-shifted absorption and fluorescence-emission maxima with respect to parental iLOV. Here, we describe the experimental characterization of this variant, which, surprisingly contrary to the theoretical prediction, shows blue-shifted absorption and fluorescence-emission maxima. Using molecular dynamics (MD) simulations and QM/MM calculations, the molecular basis for the contradictory theoretical and experimental results is presented. Essentially, our computational analysis suggests that, in the Q489K variant, two possible side-chain conformers exist: (i) a least populated conformer K489in forming a hydrogen bond with the O4 atom of FMN chromophore and (ii) a most populated conformer K489out with the side-chain amino group flipped away from the FMN chromophore forming a new hydrogen bond with the backbone oxygen of G487. QM/MM calculated spectra of the K489out conformer are blue-shifted compared to the calculated spectra of parental iLOV, which is in accordance with experimental data. This suggests that the change in the conformation of K489 from K498in to K489out accounts for the change in the direction of the spectral shift from red to blue, thus reconciling theory and experiment.

Purification and simultaneous immobilization of Arabidopsis thaliana hydroxynitrile lyase using a family 2 carbohydrate-binding module.

Tedious, time- and labor-intensive protein purification and immobilization procedures still represent a major bottleneck limiting the widespread application of enzymes in synthetic chemistry and industry. We here exemplify a simple strategy for the direct site-specific immobilization of proteins from crude cell extracts by fusion of a family 2 carbohydrate-binding module (CBM) derived from the exoglucanase/xylanase Cex from Cellulomonas fimi to a target enzyme. By employing a tripartite fusion protein consisting of the CBM, a flavin-based fluorescent protein (FbFP), and the Arabidopsis thaliana hydroxynitrile lyase (AtHNL), binding to cellulosic carrier materials can easily be monitored via FbFP fluorescence. Adsorption properties (kinetics and quantities) were studied for commercially available Avicel PH-101 and regenerated amorphous cellulose (RAC) derived from Avicel. The resulting immobilizates showed similar activities as the wild-type enzyme but displayed increased stability in the weakly acidic pH range. Finally, Avicel, RAC and cellulose acetate (CA) preparations were used for the synthesis of (R)-mandelonitrile in micro-aqueous methyl tert-butyl ether (MTBE) demonstrating the applicability and stability of the immobilizates for biotransformations in both aqueous and organic reaction systems.

Fusion of a flavin-based fluorescent protein to hydroxynitrile lyase from Arabidopsis thaliana improves enzyme stability.

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.

Synthesis of chiral cyanohydrins by recombinant Escherichia coli cells in a micro-aqueous reaction system.

Synthesis of chiral cyanohydrins is performed in a monophasic micro-aqueous reaction system using whole recombinant Escherichia coli cells expressing the Arabidopsis thaliana hydroxynitrile lyase (AtHNL). Microscopy studies employing a fusion of AtHNL with a flavin-based fluorescent protein (FbFP) reveal that the cells remain intact in the reaction system.