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Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized Chlorella variabilis FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer.

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Fatty acid photodecarboxylase (FAP) is one of the three enzymes that require light for their catalytic cycle (photoenzymes). FAP has been first identified in the green microalga Chlorella variabilis NC64A and belongs an algae-specific subgroup of the glucose-methanol-choline oxidoreductase family. While the FAP from Chlorella and its Chlamydomonas reinhardtii homolog CrFAP have demonstrated in vitro activity, their activity and physiological function have not been studied in vivo. Besides, the conservation of FAP activity beyond green microalgae remains hypothetical. Here, using a Chlamydomonas FAP knockout line (fap), we show that CrFAP is responsible for the formation of 7-heptadecene, the only hydrocarbon present in this alga. We further show that CrFAP is associated to the thylakoids and that 90% of 7-heptadecene is recovered in this cell fraction. In the fap mutant, photosynthesis activity was not affected under standard growth conditions but was reduced after cold acclimation. A phylogenetic analysis including sequences from Tara Ocean identified almost 200 putative FAPs and indicated that FAP was acquired early after primary endosymbiosis. Within Bikonta, FAP was kept in photosynthetic secondary endosymbiosis lineages but absent in those that lost the plastid. Characterization of recombinant FAPs from various algal genera (Nannochloropsis, Ectocarpus, Galdieria, Chondrus) provided experimental evidence that FAP activity is conserved in red and brown algae and is not limited to unicellular species. These results thus indicate that FAP has been conserved during evolution of most algal lineages when photosynthesis was kept and suggest that its function is linked to photosynthetic membranes.One sentence summaryFAP is present in thylakoids and conserved beyond green algae.

The aim of this study was to design a convenient, specific, sensitive, and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was extracted from Parinari glaberrimum seed kernels and the purified TAGs were used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluorescent because more than half of the fatty acids from Parinari oil are known to contain 9,11,13,15-octadecatetraenoic acid (parinaric acid) in its esterified form. The presence of detergents (sodium taurodeoxycholate, CHAPS, Sulfobetaine SB12, Tween ® 20, Brij ® 35, Dobanol ® , n-dodecylglucoside) above their critical micellar concentration dramatically increases the fluorescence of the parinaric acid released by various lipases. This increase in the fluorescence intensity is linear with time and proportional to the amount of lipase added. This new method, performed under non-oxidative conditions, was applied successfully to detecting low lipase levels in crude protein extracts from plant seeds and could be scaled down to microtiterplate measurements. Quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions (pH 8). Lipase activity can also be assayed in acidic media (pH 5) using human gastric lipase. This simple and continuous assay is compatible with a high sample throughput and might be applied to detecting true lipase activities in various biological samples. -Beisson, F., N. Ferté, J. Nari, G. Noat, V. Arondel, and R. Verger. Use of naturally fluorescent triacylglycerols from Parinari glaberrimum to detect low lipase activities from Arabidopsis thaliana seedlings.

Ongoing climate change is driving the search for renewable and carbon-neutral alternatives to fossil fuels. Photocatalytic conversion of fatty acids to hydrocarbons by fatty acid photodecarboxylase (FAP) represents a promising route to green fuels. However, the alleged low activity of FAP on C2 to C12 fatty acids seemed to preclude the use for synthesis of gasoline-range hydrocarbons. Here, we reveal that Chlorella variabilis FAP ( Cv FAP) can convert n -octanoic acid in vitro four times faster than n -hexadecanoic acid, its best substrate reported to date. In vivo, this translates into a Cv FAP-based production rate over 10-fold higher for n -heptane than for n -pentadecane. Time-resolved spectroscopy and molecular modeling demonstrate that Cv FAP’s high catalytic activity on n -octanoic acid is, in part, due to an autocatalytic effect of its n -heptane product, which fills the rest of the binding pocket. These results represent an important step toward a bio-based and light-driven production of gasoline-like hydrocarbons.

are able to form extraordinarily stable vesicular membranes against a number of chemical, physical and mechanical stressors. In this study, we demonstrated that PLFE can also form free-standing ''planar'' membranes on micro-pores (~100 micrometer) of polydimethylsiloxane (PDMS) thin films embedded in printed circuit board (PCB)-based fluidics. Using electrochemical impedance spectroscopy (EIS), we found that the dielectric properties of PLFE planar membranes suspended on the PDMS films are distinctly different from those obtained from diester lipid and triblock copolymer membranes. In addition to resistance (R) and capacitance (C) that were seen in all the membranes examined, PLFE planar membranes showed an inductance (L) component. Furthermore, PLFE planar membranes displayed a relatively large membrane resistance, suggesting that, among the membranes examined, PLFE planar membrane would be a better matrix for studying channel proteins and transmembrane events. PLFE planar membranes also exhibited a sharp decrease in phase angle with the frequency of the input AC signal at~1 MHz, which could be utilized to develop sensors for monitoring PLFE membrane integrity in fluidics. Since the stability of free-standing planar lipid membranes increases with increasing membrane packing tightness and PLFE lipid membranes are more tightly packed than those made of diester lipids, PLFE free-standing planar membranes are expected to be considerably stable. All these salient features make PLFE planar membranes particularly attractive for model studies of channel proteins and transmembrane events and for high-throughput drug screening. Terrestrial plants regulate environmental interactions via insoluble polymers assembled in their epidermal and/or peridermal cell walls. The plant cuticles with waterproofing and antimicrobial capabilities represent a unique class of biological assemblies composed of ester-linked and insoluble constituents such as biopolyester cutins, lipid waxes, and chemically recalcitrant cutans. Solid-state nuclear magnetic resonance (NMR) offers a powerful technique to probe the structural and dynamical properties of these geochemically important and structurally amorphous biological systems that can also motivate the engineering of water-resistant bioinspired materials from renewable sources. Based on key genes and biosynthetic pathways identified in Arabidopsis thaliana leaf cuticles, our current solid-state NMR study delineates compositional variations and multiple-timescale (ms-ns) dynamics for several genetically tailored and insoluble plant cuticle systems, linking macromolecular organization with protective performance and operational design. 2454-Pos Board B591Injectable Reverse Thermal Gel Biopolymers may Act as an Extracellular Matrix and Cell Vehicle for Cardiac Tissue Engineering Background: Recent investigations demonstrated that tissue engineering represents a promising strategy to repair diseased hearts. We hypothesized that temperature-responsive materials could be developed as extracellular ma...