Fluorescent reporter proteins such as green fluorescent protein are valuable noninvasive molecular tools for in vivo real-time imaging of living specimens. However, their use is generally restricted to aerobic systems, as the formation of their chromophores strictly requires oxygen. Starting with blue-light photoreceptors from Bacillus subtilis and Pseudomonas putida that contain light-oxygen-voltage-sensing domains, we engineered flavin mononucleotide-based fluorescent proteins that can be used as fluorescent reporters in both aerobic and anaerobic biological systems.

In this study photophysical characteristics of LOV-based fluorescent proteins which are essential for analytic methods as well as imaging approaches have been comparatively analyzed in detail.

We previously characterized a LOV protein PpSB2-LOV, present in the common soil bacterium Pseudomonas putida, that exhibits a plant phototropin LOV-like photochemistry [Krauss, U., Losi, A., Gartner, W., Jaeger, K. E., and Eggert, T. (2005) Phys. Chem. Chem. Phys. 7, 2804-2811]. Now, we have identified a second LOV homologue, PpSB1-LOV, found in the same organism with approximately 66% identical amino acids. Both proteins consist of a conserved LOV core flanked by short N- and C-terminal extensions but lack a fused effector domain. Although both proteins are highly similar in sequence, they display drastically different dark recovery kinetics. At 20 degrees C, PpSB2-LOV reverts with an average time constant of 137 s from the photoequilibrium to the dark state, whereas PpSB1-LOV exhibits an average dark recovery time constant of 1.48 x 10(5) s. Irrespective of the significant differences in their dark recovery behavior, both proteins showed nearly identical kinetics for the photochemically induced adduct formation. In order to elucidate the structural and mechanistic basis of these extremely different dark recovery time constants, we performed a mutational analysis. Six amino acids in a distance of up to 6 A from the flavin chromophore, which differ between the two proteins, were identified and interchanged by site-directed mutagenesis. The amino acid substitution R66I located near the FMN phosphate in LOV domains was identified in PpSB1-LOV to accelerate the dark recovery by 2 orders of magnitude. Vice versa, the corresponding substitution I66R slowed down the dark recovery in PpSB2-LOV by a factor of 10. Interestingly, the interchange of the C-terminal extensions between the two proteins also had a pronounced effect on the dark recovery time constants, thus highlighting a coupling of these protein regions to the chromophore binding pocket.

The increasing number of biocatalytic reactions implemented in chemical synthesis routes raises the urgent need for large amounts of enzymes. Hence, new generic methods are required for their simple and cost‐efficient production. Here, we describe a generally applicable method based on the production of catalytically active inclusion bodies (CatIBs). CatIBs represent a promising new form of biologically produced, carrier‐free, biodegradable enzyme immobilizate. CatIBs are produced in Escherichia coli by expression of a gene fusion consisting of a coiled‐coil domain and a target enzyme. Employing this strategy, the lipase A of Bacillus subtilis (BsLA), the hydroxynitrile lyase of Arabidopsis thaliana (AtHNL), and the 2‐succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate synthase MenD of E. coli (EcMenD) were successfully produced as CatIBs and used in aqueous and micro‐aqueous organic solvent based reaction systems, showing excellent stability and recyclability.

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The Bacillus subtilis protein YtvA is related to plant phototropins in that it senses UVA-blue-light by means of the flavin binding LOV domain, linked to a nucleotide-binding STAS domain. The structural basis for interdomain interactions and functional regulation are not known. Here we report the conformational analysis of three YtvA constructs, by means of size exclusion chromatography, circular dichroism (CD) and molecular docking simulations. The isolated YtvA-LOV domain (YLOV, aa 25-126) has a strong tendency to dimerize, prevented in full-length YtvA, but still observed in YLOV carrying the N-terminal extension (N-YLOV, aa 1-126). The analysis of CD data shows that both the N-terminal cap and the linker region (aa 127-147) between the LOV and the STAS domain are helical and that the central beta-scaffold is distorted in the LOV domains dimers. The involvement of the central beta-scaffold in dimerization is supported by docking simulation of the YLOV dimer and the importance of this region is highlighted by light-induced conformational changes, emerging from the CD data analysis. In YtvA, the beta-strand fraction is notably less distorted and distinct light-driven changes in the loops/turn fraction are detected. The data uncover a common surface for LOV-LOV and intraprotein interaction, involving the central beta-scaffold, and offer hints to investigate the molecular basis of light-activation and regulation in LOV proteins.

In nature, enzymatic reaction cascades, i.e., realized in metabolic networks, operate with unprecedented efficacy, with the reactions often being spatially and temporally orchestrated. The principle of "learning from nature" has in recent years inspired the setup of synthetic reaction cascades combining biocatalytic reaction steps to artificial cascades. Hereby, the spatial organization of multiple enzymes, e.g., by coimmobilization, remains a challenging task, as currently no generic principles are available that work for every enzyme. We here present a tunable, genetically programmed coimmobilization strategy that relies on the fusion of a coiled-coil domain as aggregation inducing-tag, resulting in the formation of catalytically active inclusion body coimmobilizates (Co-CatIBs). Coexpression and coimmobilization was proven using two fluorescent proteins, and the strategy was subsequently extended to two enzymes, which enabled the realization of an integrated enzymatic two-step cascade for the production of (1 R,2 R)-1-phenylpropane-1,2-diol (PPD), a precursor of the calicum channel blocker diltiazem. In particular, the easy production and preparation of Co-CatIBs, readily yielding a biologically produced enzyme immobilizate renders the here presented strategy an interesting alternative to existing cascade immobilization techniques.

Plants and fungi respond to environmental light stimuli via the action of different photoreceptor modules. One such class, responding to the blue region of light, is constituted by photoreceptors containing so-called light-oxygen-voltage (LOV) domains as sensor modules. Four major LOV families are currently identified in eukaryotes: (i) the plant phototropins, regulating various physiological effects such as phototropism, chloroplast relocation, and stomatal opening; (ii) the aureochromes, mediating photomorphogenesis in photosynthetic stramenopile algae; (iii) the plant circadian photoreceptors of the zeitlupe (ZTL)/adagio (ADO)/flavinbinding Kelch repeat F-box protein 1 (FKF1) family; and (iv) the fungal circadian photoreceptors white-collar 1 (WC-1). Blue-light-sensitive LOV signaling modules are also widespread throughout the prokaryotic world, and physiological responses mediated by bacterial LOV photoreceptors were recently reported. Thus, the question arises as to the evolutionary relationship between the pro-and eukaryotic LOV photoreceptor systems. We used Bayesian and maximum-likelihood tree reconstruction methods to infer evolutionary scenarios that might have led to the widespread appearance of LOV domains among the pro-and eukaryotes. The phylogenetic study presented here suggests a bacterial origin for the LOV domains of the four major eukaryotic LOV photoreceptor families, whereas the LOV sensor domains were most likely recruited from the bacteria in the course of plastid and mitochondrial endosymbiosis.A plant family of photoreceptors, the phototropins, containing so-called light-oxygen-voltage (LOV) domains as the bluelight-sensitive signaling switches (12), were previously identified as the key modulators of a variety of plant blue-light responses, including plant phototropism (23), chloroplast relocation, and stomatal and leaf opening (25,39). A second family (ZTL/ADO/FKF1 family) of eukaryotic LOV domaincontaining proteins is constituted by the zeitlupe (ZTL) and the flavin-binding Kelch repeat F-box (FKF1) proteins. This family was found to play a primary role in the photocontrol of flowering and in the light-dependent regulation of the circadian period in plants (37, 53). Phototropins and ZTLs together with the fungal white-collar 1 (WC-1) proteins of, e.g., Neurospora crassa (3), which are involved in the blue-light dependent control of fungal circadian responses (19), constitute the three major eukaryotic LOV families in plants and fungi. A more recently discovered fourth family of LOV domain-containing blue-light receptors, the so-called aureochromes (58), seem to be restricted to stramenopile algae such as Vaucheria frigida and some marine diatoms (e.g., Thalassiosira pseudonana). Recently, the presence of LOV domain signaling modules was also predicted for animals, including humans. This functional assignment was solely based on sequence similarity to known LOV systems in plants and bacteria (62); however, experimental evidence is still missing. LOV domains are small (110 amino acids),...