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Although many organisms capture or respond to sunlight, few enzymes are known to be driven by light. Among these are DNA photolyases and the photosynthetic reaction centers. Here, we show that the microalga Chlorella variabilis NC64A harbors a photoenzyme that acts in lipid metabolism. This enzyme belongs to an algae-specific clade of the glucose-methanol-choline oxidoreductase family and catalyzes the decarboxylation of free fatty acids to n-alkanes or -alkenes in response to blue light. Crystal structure of the protein reveals a fatty acid–binding site in a hydrophobic tunnel leading to the light-capturing flavin adenine dinucleotide (FAD) cofactor. The decarboxylation is initiated through electron abstraction from the fatty acid by the photoexcited FAD with a quantum yield >80%. This photoenzyme, which we name fatty acid photodecarboxylase, may be useful in light-driven, bio-based production of hydrocarbons.

Plants adjust their photosynthetic activity to changing light conditions. A central regulation of photosynthesis depends on the xanthophyll cycle, in which the carotenoid violaxanthin is converted into zeaxanthin in strong light, thus activating the dissipation of the excess absorbed energy as heat and the scavenging of reactive oxygen species. Violaxanthin deepoxidase (VDE), the enzyme responsible for zeaxanthin synthesis, is activated by the acidification of the thylakoid lumen when photosynthetic electron transport exceeds the capacity of assimilatory reactions: at neutral pH, VDE is a soluble and inactive enzyme, whereas at acidic pH, it attaches to the thylakoid membrane where it binds its violaxanthin substrate. VDE also uses ascorbate as a cosubstrate with a pH-dependent Km that may reflect a preference for ascorbic acid. We determined the structures of the central lipocalin domain of VDE (VDEcd) at acidic and neutral pH. At neutral pH, VDEcd is monomeric with its active site occluded within a lipocalin barrel. Upon acidification, the barrel opens up and the enzyme appears as a dimer. A channel linking the two active sites of the dimer can harbor the entire carotenoid substrate and thus may permit the parallel deepoxidation of the two violaxanthin β-ionone rings, making VDE an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate.

Magnetotactic bacteria contain magnetosomes—intracellular, membrane-bounded, magnetic nanocrystals of magnetite (Fe₃O₄) or greigite (Fe₃S₄)— that cause the bacteria to swim along geomagnetic field lines. We isolated a greigite-producing magnetotactic bacterium from a brackish spring in Death Valley National Park, California, USA, strain BW-1, that is able to biomineralize greigite and magnetite depending on culture conditions. A phylogenetic comparison of BW-1 and similar uncultured greigite- and/or magnetite-producing magnetotactic bacteria from freshwater to hypersaline habitats shows that these organisms represent a previously unknown group of sulfate-reducing bacteria in the Deltaproteobacteria. Genomic analysis of BW-1 reveals the presence of two different magnetosome gene clusters, suggesting that one may be responsible for greigite biomineralization and the other for magnetite.

Enzymes are versatile catalysts in laboratories and on an industrial scale; improving their immobilization would be beneficial to broadening their applicability and ensuring their (re)use. Lipid-coated nano-magnets produced by magnetotactic bacteria are suitable for a universally applicable single-step method of enzyme immobilization. By genetically functionalizing the membrane surrounding these magnetite particles with a phosphohydrolase, we engineered an easy-to-purify, robust and recyclable biocatalyst to degrade ethyl-paraoxon, a commonly used pesticide. For this, we genetically fused the opd gene from Flavobacterium sp. ATCC 27551 encoding a paraoxonase to mamC, an abundant protein of the magnetosome membrane in Magnetospirillum magneticum AMB-1. The MamC protein acts as an anchor for the paraoxonase to the magnetosome surface, thus producing magnetic nanoparticles displaying phosphohydrolase activity. Magnetosomes functionalized with Opd were easily recovered from genetically modified AMB-1 cells: after cellular disruption with a French press, the magnetic nanoparticles are purified using a commercially available magnetic separation system. The catalytic properties of the immobilized Opd were measured on ethyl-paraoxon hydrolysis: they are comparable with the purified enzyme, with K(m) (and k(cat)) values of 58 µM (and 178 s(-1)) and 43 µM (and 314 s(-1)) for the immobilized and purified enzyme respectively. The Opd, a metalloenzyme requiring a zinc cofactor, is thus properly matured in AMB-1. The recycling of the functionalized magnetosomes was investigated and their catalytic activity proved to be stable over repeated use for pesticide degradation. In this study, we demonstrate the easy production of functionalized magnetic nanoparticles with suitably genetically modified magnetotactic bacteria that are efficient as a reusable nanobiocatalyst for pesticides bioremediation in contaminated effluents.

Under the same selection pressures, two genetically divergent populations may evolve in parallel toward the same adaptive solutions. Here, we hypothesized that magnetotaxis (i.e., magnetically guided chemotaxis) represents a key adaptation to micro-oxic habitats in aquatic sediments and that its parallel evolution homogenized the phenotypes of two evolutionary divergent clusters of freshwater spirilla. All magnetotactic bacteria affiliated to the Magnetospirillum genus (Alphaproteobacteria class) biomineralize the same magnetic particle chains and share highly similar physiological and ultrastructural features. We looked for the processes that could have contributed at shaping such an evolutionary pattern by reconciling species and gene trees using newly sequenced genomes of Magnetospirillum related bacteria. We showed that repeated horizontal gene transfers and homologous recombination of entire operons contributed to the parallel evolution of magnetotaxis. We propose that such processes could represent a more parsimonious and rapid solution for adaptation compared with independent and repeated de novo mutations, especially in the case of traits as complex as magnetotaxis involving tens of interacting proteins. Besides strengthening the idea about the importance of such a function in micro-oxic habitats, these results reinforce previous observations in experimental evolution suggesting that gene flow could alleviate clonal interference and speed up adaptation under some circumstances.

Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein.

Phytochelatin synthase (PCS) is a key enzyme for heavy-metal detoxification in plants. PCS catalyzes the production of glutathione (GSH)-derived peptides (called phytochelatins or PCs) that bind heavy-metal ions before vacuolar sequestration. The enzyme can also hydrolyze GSH and GS-conjugated xenobiotics. In the cyanobacterium Nostoc, the enzyme (NsPCS) contains only the catalytic domain of the eukaryotic synthase and can act as a GSH hydrolase and weakly as a peptide ligase. The crystal structure of NsPCS in its native form solved at a 2.0-Å resolution shows that NsPCS is a dimer that belongs to the papain superfamily of cysteine proteases, with a conserved catalytic machinery. Moreover, the structure of the protein solved as a complex with GSH at a 1.4-Å resolution reveals a γ-glutamyl cysteine acyl-enzyme intermediate stabilized in a cavity of the protein adjacent to a second putative GSH binding site. GSH hydrolase and PCS activities of the enzyme are discussed in the light of both structures.

Mutualistic symbioses are often a source of evolutionary innovation and drivers of biological diversification 1 . Widely distributed in the microbial world, particularly in anoxic settings 2 , 3 , they often rely on metabolic exchanges and syntrophy 2 , 4 . Here, we report a mutualistic symbiosis observed in marine anoxic sediments between excavate protists (Symbiontida, Euglenozoa) 5 and ectosymbiotic Deltaproteobacteria biomineralizing ferrimagnetic nanoparticles. Light and electron microscopy observations as well as genomic data support a multi-layered mutualism based on collective magnetotactic motility with division of labour and interspecies hydrogen-transfer-based syntrophy 6 . The guided motility of the consortia along the geomagnetic field is allowed by the magnetic moment of the non-motile ectosymbiotic bacteria combined with the protist motor activity, which is a unique example of eukaryotic magnetoreception 7 acquired by symbiosis. The nearly complete deltaproteobacterial genome assembled from a single consortium contains a full magnetosome gene set 8 , but shows signs of reduction, with the probable loss of flagellar genes. Based on the metabolic gene content, the ectosymbiotic bacteria are anaerobic sulfate-reducing chemolithoautotrophs that likely reduce sulfate with hydrogen produced by hydrogenosome-like organelles 6 underlying the plasma membrane of the protist. In addition to being necessary hydrogen sinks, ectosymbionts may provide organics to the protist by diffusion and predation, as shown by magnetosome-containing digestive vacuoles. Phylogenetic analyses of 16S and 18S ribosomal RNA genes from magnetotactic consortia in marine sediments across the Northern and Southern hemispheres indicate a host–ectosymbiont specificity and co-evolution. This suggests a historical acquisition of magnetoreception by a euglenozoan ancestor from Deltaproteobacteria followed by subsequent diversification. It also supports the cosmopolitan nature of this type of symbiosis in marine anoxic sediments. Here, the authors identify a mutualistic symbiosis between a non-motile, magnetic deltaproteobacterium and a protist, resulting in eukaryotic magnetoreception in marine anoxic sediments.

Magnetotactic bacteria are able to swim navigating along geomagnetic field lines. They synthesize ferromagnetic nanocrystals that are embedded in cytoplasmic membrane invaginations forming magnetosomes. Regularly aligned in the cytoplasm along cytoskeleton filaments, the magnetosome chain effectively forms a compass needle bestowing on bacteria their magnetotactic behaviour. A large genomic island, conserved among magnetotactic bacteria, contains the genes potentially involved in magnetosome formation. One of the genes, mamK has been described as encoding a prokaryotic actin-like protein which when it polymerizes forms in the cytoplasm filamentous structures that provide the scaffold for magnetosome alignment. Here, we have identified a series of genes highly similar to the mam genes in the genome of Magnetospirillum magneticum AMB-1. The newly annotated genes are clustered in a genomic islet distinct and distant from the known magnetosome genomic island and most probably acquired by lateral gene transfer rather than duplication. We focused on a mamK-like gene whose product shares 54.5% identity with the actin-like MamK. Filament bundles of polymerized MamK-like protein were observed in vitro with electron microscopy and in vivo in E. coli cells expressing MamK-like-Venus fusions by fluorescence microscopy. In addition, we demonstrate that mamK-like is transcribed in AMB-1 wild-type and DeltamamK mutant cells and that the actin-like filamentous structures observed in the DeltamamK strain are probably MamK-like polymers. Thus MamK-like is a new member of the prokaryotic actin-like family. This is the first evidence of a functional mam gene encoded outside the magnetosome genomic island.

Doc number: 28 Abstract Background: YedY, a molybdoenzyme belonging to the sulfite oxidase family, is found in most Gram-negative bacteria. It contains a twin-arginine signal sequence that is cleaved after its translocation into the periplasm. Despite a weak reductase activity with substrates such as dimethyl sulfoxide or trimethylamine N-oxide, its natural substrate and its role in the cell remain unknown. Although sequence conservation of the YedY family displays a strictly conserved hydrophobic C-terminal residue, all known studies on Escherichia coli YedY have been performed with an enzyme containing a 6 histidine-tag at the C-terminus which could hamper enzyme activity. Results: In this study, we demonstrate that the tag fused to the C-terminus of Rhodobacter sphaeroides YedY is detrimental to the enzyme's reductase activity and results in an eight-fold decrease in catalytic efficiency. Nonetheless this C-terminal tag does not influence the properties of the molybdenum active site, as assayed by EPR spectroscopy. When a cleavable His-tag was fused to the N-terminus of the mature enzyme in the absence of the signal sequence, YedY was expressed and folded with its cofactor. However, when the signal sequence was added upstream of the N-ter tag, the amount of enzyme produced was approximately ten-fold higher. Conclusion : Our study thus underscores the risk of using a C-terminus tagged enzyme while studying YedY, and presents an alternative strategy to express signal sequence-containing enzymes with an N-terminal tag. It brings new insights into molybdoenzyme maturation in R. sphaeroides showing that for some enzymes, maturation can occur in the absence of the signal sequence but that its presence is required for high expression of active enzyme.