Calculation of the Geometries and Infrared Spectra of the Stacked Cofactor Flavin Adenine Dinucleotide (FAD) as the Prerequisite for Studies of Light-Triggered Proton and Electron Transfer

Kieninger M, Ventura ON, Kottke T. Calculation of the Geometries and Infrared Spectra of the Stacked Cofactor Flavin Adenine Dinucleotide (FAD) as the Prerequisite for Studies of Light-Triggered Proton and Electron Transfer. Biomolecules. 2020;10(4): 573.Flavin cofactors, like flavin adenine dinucleotide (FAD), are important electron shuttles in living systems. They catalyze a wide range of one- or two-electron redox reactions. Experimental investigations include UV-vis as well as infrared spectroscopy. FAD in aqueous solution exhibits a significantly shorter excited state lifetime than its analog, the flavin mononucleotide. This finding is explained by the presence of a “stacked” FAD conformation, in which isoalloxazine and adenine moieties form a π-complex. Stacking of the isoalloxazine and adenine rings should have an influence on the frequency of the vibrational modes. Density functional theory (DFT) studies of the closed form of FAD in microsolvation (explicit water) were used to reproduce the experimental infrared spectra, substantiating the prevalence of the stacked geometry of FAD in aqueous surroundings. It could be shown that the existence of the closed structure in FAD can be narrowed down to the presence of only a single water molecule between the third hydroxyl group (of the ribityl chain) and the N7 in the adenine ring of FAD

Arguments for an additional long-lived intermediate in the photocycle of the full-length aureochrome 1c receptor: A time-resolved small-angle X-ray scattering study

Bannister S, Böhm E, Zinn T, Hellweg T, Kottke T. Arguments for an additional long-lived intermediate in the photocycle of the full-length aureochrome 1c receptor: A time-resolved small-angle X-ray scattering study. Structural Dynamics. 2019;6(3): 34701.Aureochromes (AUREO) act as blue-light photoreceptors in algae. They consist of a light-, oxygen-, voltage-sensitive (LOV) domain and a DNA-binding basic region/leucine zipper. Illumination of the flavin cofactor in LOV leads to the formation of an adduct, followed by global structural changes. Here, we first applied UV/vis spectroscopy to characterize the photocycle of full-length aureochrome 1c (PtAUREO1c) from the diatom Phaeodactylum tricornutum. With a time constant of 850 s and a quantum yield of 23%, PtAUREO1c reveals a faster recovery time and a much lower sensitivity toward light than PtAUREO1a, pointing to its role as a high light sensor in vivo. UV/vis spectroscopy offers details on the local recovery of the flavin chromophore. However, kinetic information on the global structural recovery of full-length AUREO or any other multidomain LOV protein is missing. This information is essential not least for the photoreceptors' applications as optogenetic devices. Therefore, we established a procedure to apply small-angle X-ray scattering on PtAUREO1c in a time-resolved manner employing an in-house setup. In combination with UV/vis spectroscopy under similar conditions, we revealed a discrepancy between the recovery of the global protein structure and the adduct lifetime. Accordingly, we propose to supplement the photocycle by an intermediate state (I447), which decays with a time constant of about 800 s and prolongs the lifetime of the signaling state

Boreal pollen contain ice-nucleating as well as ice-binding ‘antifreeze’ polysaccharides

Dreischmeier K, Budke C, Wiehemeier L, Kottke T, Koop T. Boreal pollen contain ice-nucleating as well as ice-binding ‘antifreeze’ polysaccharides. Scientific Reports. 2017;7(1): 41890.Ice nucleation and growth is an important and widespread environmental process. Accordingly, nature has developed means to either promote or inhibit ice crystal formation, for example ice-nucleating proteins in bacteria or ice-binding antifreeze proteins in polar fish. Recently, it was found that birch pollen release ice-nucleating macromolecules when suspended in water. Here we show that birch pollen washing water exhibits also ice-binding properties such as ice shaping and ice recrystallization inhibition, similar to antifreeze proteins. We present spectroscopic evidence that both the ice-nucleating as well as the ice-binding molecules are polysaccharides bearing carboxylate groups. The spectra suggest that both polysaccharides consist of very similar chemical moieties, but centrifugal filtration indicates differences in molecular size: ice nucleation occurs only in the supernatant of a 100 kDa filter, while ice shaping is strongly enhanced in the filtrate. This finding may suggest that the larger ice-nucleating polysaccharides consist of clusters of the smaller ice-binding polysaccharides, or that the latter are fragments of the ice-nucleating polysaccharides. Finally, similar polysaccharides released from pine and alder pollen also display both ice-nucleating as well as ice-binding ability, suggesting a common mechanism of interaction with ice among several boreal pollen with implications for atmospheric processes and antifreeze protection

Activation of recombinantly expressed l-Amino Acid Oxidase from Rhizoctonia solani by Sodium Dodecyl Sulfate

Hahn K, Hertle Y, Bloess S, Kottke T, Hellweg T, Fischer von Mollard G. Activation of recombinantly expressed l-Amino Acid Oxidase from Rhizoctonia solani by Sodium Dodecyl Sulfate. Molecules. 2017;22(12): 2272.l-Amino acid oxidases (l-AAO) catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids. The non-covalently bound cofactor FAD is reoxidized by oxygen under formation of hydrogen peroxide. We expressed an active l-AAO from the fungus Rhizoctonia solani as a fusion protein in E. coli. Treatment with small amounts of the detergent sodium dodecyl sulfate (SDS) stimulated the activity of the enzyme strongly. Here, we investigated whether other detergents and amphiphilic molecules activate 9His-rsLAAO1. We found that 9His-rsLAAO1 was also activated by sodium tetradecyl sulfate. Other detergents and fatty acids were not effective. Moreover, effects of SDS on the oligomerization state and the protein structure were analyzed. Native and SDS-activated 9His-rsLAAO1 behaved as dimers by size-exclusion chromatography. SDS treatment induced an increase in hydrodynamic radius as observed by size-exclusion chromatography and dynamic light scattering. The activated enzyme showed accelerated thermal inactivation and an exposure of additional protease sites. Changes in tryptophan fluorescence point to a more hydrophilic environment. Moreover, FAD fluorescence increased and a lower concentration of sulfites was sufficient to form adducts with FAD. Taken together, these data point towards a more open conformation of SDS-activated l-amino acid oxidase facilitating access to the active site

A flavin-dependent halogenase from metagenomic analysis prefers bromination over chlorination

Neubauer P, Widmann C, Wibberg D, et al. A flavin-dependent halogenase from metagenomic analysis prefers bromination over chlorination. PLoS ONE. 2018;13(5): e0196797.Flavin-dependent halogenases catalyse halogenation of aromatic compounds. In most cases, this reaction proceeds with high regioselectivity and requires only the presence of FADH2, oxygen, and halide salts. Since marine habitats contain high concentrations of halides, organisms populating the oceans might be valuable sources of yet undiscovered halogenases. A new Hidden-Markov-Model (HMM) based on the PFAM tryptophan halogenase model was used for the analysis of marine metagenomes. Eleven metagenomes were screened leading to the identification of 254 complete or partial putative flavin-dependent halogenase genes. One predicted halogenase gene (brvH) was selected, codon optimised for E. coli, and overexpressed. Substrate screening revealed that this enzyme represents an active flavin-dependent halogenase able to convert indole to 3-bromoindole. Remarkably, bromination prevails also in a large excess of chloride. The BrvH crystal structure is very similar to that of tryptophan halogenases but reveals a substrate binding site that is open to the solvent instead of being covered by a loop

Phosphorus and nitrogen starvation reveal life-cycle specific responses in the metabolome of Emiliania huxleyi (Haptophyta)

The coccolithophore Emiliania huxleyi is a microalga with biogeochemical and biotechnological relevance, due to its high abundance in the ocean and its ability to form intricate calcium carbonate structures. Depletion of macronutrients in oceanic waters is very common and will likely enhance with advancing climate change. We present the first comprehensive metabolome study analyzing the effect of phosphorus (P) and nitrogen (N) starvation on the diploid and haploid life-cycle stage, applying various metabolome analysis methods to gain new insights in intracellular mechanisms to cope with nutrient starvation. P-starvation led to an accumulation of many generic and especially N-rich metabolites, including lipids, osmolytes, and pigments. This suggests that P-starvation primarily arrests cell-cycling due to lacking P for nucleic acid synthesis, but that enzymatic functionality is widely preserved. Also, the de-epoxidation ratio of the xanthophyll cycle was upregulated in the diploid stage under P-starvation, indicating increased nonphotochemical quenching, a response typically observed under high light stress. In contrast, N-starvation resulted in a decrease of most central metabolites, also P-containing ones, especially in the diploid stage, indicating that most enzymatic functionality ceased. The two investigated nutrient starvation conditions caused significantly different responses, contrary to previous assumptions derived from transcriptomic studies. Data highlight that instantaneous biochemical flux is a more dominant driver of the metabolome than the transcriptomically rearranged pathway patterns. Due to the fundamental nature of the observed responses it may be speculated that microalgae with similar nutrient requirements can cope better with P-starvation than with N-starvation

The World of Algae Reveals a Broad Variety of Cryptochrome Properties and Functions

Algae are photosynthetic eukaryotic (micro-)organisms, lacking roots, leaves, and other organs that are typical for land plants. They live in freshwater, marine, or terrestrial habitats. Together with the cyanobacteria they contribute to about half of global carbon fixation. As primary producers, they are at the basis of many food webs and they are involved in biogeochemical processes. Algae are evolutionarily distinct and are derived either by primary (e.g., green and red algae) or secondary endosymbiosis (e.g., diatoms, dinoflagellates, and brown algae). Light is a key abiotic factor needed to maintain the fitness of algae as it delivers energy for photosynthesis, regulates algal cell- and life cycles, and entrains their biological clocks. However, excess light can also be harmful, especially in the ultraviolet range. Among the variety of receptors perceiving light information, the cryptochromes originally evolved as UV-A and blue-light receptors and have been found in all studied algal genomes so far. Yet, the classification, biophysical properties, wavelength range of absorbance, and biological functions of cryptochromes are remarkably diverse among algal species, especially when compared to cryptochromes from land plants or animals

UNEXPECTED DIVERSITY OF CRYPTOCHROME PHOTORECEPTORS FROM ALGAE

Kottke T. UNEXPECTED DIVERSITY OF CRYPTOCHROME PHOTORECEPTORS FROM ALGAE. European Journal of Phycology. 2015;50:63-63

Spektroskopische Aufklärung des Photozyklus der LOV1-Domäne eines Phot-Blaulichtrezeptors

Phot-Proteine steuern in ihrer Funktion als sensorische Blaulichtrezeptoren die Anpassung von Organismen an die Lichtverhältnisse in der Umgebung. In dieser Arbeit wurde der Photozyklus der lichtempfindlichen LOV1-Domäne aus dem Phot-Protein der Grünalge Chlamydomonas reinhardtii mit Hilfe der transienten Absorptionsspektroskopie untersucht. Die LOV1-Domäne enthält ein Flavin-Mononucleotid als Chromophor. Bei Belichtung geht der Chromophor zunächst in den angeregten Triplettzustand über, der mit zwei Zeitkonstanten von 800 Nanosekunden und 4 Mikrosekunden abklingt. Diese lassen sich einer effizienten Reaktion in das Photoprodukt zuordnen, das sich bei der Reaktion des Triplettzustands mit dem benachbarten Cystein 57 bildet. Das Absorptionsspektrum des Photoprodukts zeigt einen für Flavin-C(4a)-Thiol-Addukte charakteristischen Verlauf mit einem Maximum bei 390 nm. Das Addukt ist nur metastabil und reagiert mit einer Zeitkonstante von 200 s wieder in den Dunkelzustand zurück. Die Kinetik der Rückreaktion hängt stark von den Umgebungsbedingungen wie pH-Wert und Salzkonzentration der Lösung ab. Es wurden deutliche Hinweise darauf gefunden, dass die Rückreaktion auch photochemisch induziert abläuft. Um den Mechanismus der Adduktbildung aufzuklären, wurde in einer Mutante das reaktive Cystein durch ein Methionin ausgetauscht. In der Photoreaktion der C57M-Mutante bildet sich aus einer Reaktion der terminalen Methylgruppe des Methionins mit dem Flavin ein N(5)-Addukt. Dieses Addukt wird in einer thermischen Folgereaktion zu einem Neutralradikal des Flavins oxidiert. Das Produkt enthält eine kovalente Bindung zwischen Chromophor und Apoprotein und ist daher selbst unter aeroben und denaturierenden Bedingungen stabil. Damit unterscheidet es sich deutlich von anderen Flavoproteinradikalen. In der C57M-Mutante kann ein ionischer Mechanismus der Adduktbildung über eine Deprotonierung des Methionins und eine Protonierung des FMN-Triplettzustands weitgehend ausgeschlossen werden. Weitaus plausibler ist hier ein radikalischer Mechanismus, der auch auf die Reaktion des Wildtyps übertragen werden kann

A quantum cascade laser setup for studying irreversible photoreactions in H2O with nanosecond resolution and microlitre consumption

Klocke J, Kottke T. A quantum cascade laser setup for studying irreversible photoreactions in H2O with nanosecond resolution and microlitre consumption. Physical Chemistry Chemical Physics. 2020;22(45):26459-26467.Flavin photoreduction in H2O is elucidated by developing a quantum cascade laser setup for time-resolved infrared spectroscopy on irreversible reactions.</p