A promising enzyme for green chemistry in Science
The functioning of an enzyme, which could be used to produce biofuels and for green chemistry, has been deciphered. This result has mobilized an international team of scientists with an important contribution from the Laboratory of Optics and Biosciences. This discovery is published in Science on April 9, 2021.
An international consortium, including many researchers from CEA, CNRS, Inserm, École Polytechnique and the universities of Grenoble Alpes, Paris-Saclay and Aix-Marseille, as well as a Max Planck Institute in Germany and Stanford's Linear Accelerator Center (SLAC), have deciphered the working mechanisms of the FAP enzyme - for Fatty Acid Photodecarboxylase - naturally present in microscopic algae such as chlorella. This enzyme had been identified in 2017 to be able, with the help of light energy, of forming hydrocarbons from fatty acids produced by these microalgae. To achieve this new result published in Science on April 9, 2021, the research teams employed a complete experimental and theoretical panoply.
Studying ultra-fast phenomena
Due to the fact that their reaction is triggered by light, photoenzymes give access to ultra-fast phenomena taking place during enzymatic reactions. The FAP enzyme therefore also represents a unique opportunity to understand in detail a chemical reaction taking place in living organisms.
More precisely, in this work, the researchers show that when the enzyme is illuminated and absorbs a photon, an electron is stripped off in 300 picoseconds (1 picosecond = one thousandth of a billionth of a second) from the fatty acid produced by the algae.
This fatty acid is then dissociated into a hydrocarbon precursor and carbon dioxide (CO2). Most of the produced CO2 is then converted within 100 nanoseconds to bicarbonate (HCO3-) within the enzyme. The activity of the enzyme requires light, but it is not inhibited by the pigments of photosynthesis: embedded in the enzyme, the flavin molecule, which absorbs the photon, is bent. This spatial configuration, called "conformation", shifts the absorption spectrum of the molecule towards the red, so that it uses photons not exploited for the photosynthetic activity of the microalgae.
The LOB at the heart of the action
At the Laboratory of Optics and Biosciences (LOB*), Marten Vos, CNRS research director, came into contact with researchers at CEA Cadarache and Saclay who discovered this enzyme in 2017 and had been working on it since. A real teamwork was then initiated with Damien Sorigué, researcher at CEA and first author of this publication.
The LOB researchers, engineers, PhD student and intern worked on the one hand on visible spectroscopy on the femtosecond-picosecond scale to follow the photoreduction kinetics of the flavin and on the other hand on multiscale infrared spectroscopy (covering a time range extending over more than eight orders of magnitude) with a high spectral resolution to follow the formation and the fate of the CO2 product within the enzyme. The latter approach combines two original techniques developed at LOB.
First, ADASOPS (Arbitrary-Detuning ASynchronous OPtical Sampling) which allows to cover a very large temporal range using two independent femtosecond lasers, a unique technique patented by LOB. This tool, the result of 10 years of work, has mobilized the complementary skills of Laura Roby Antonucci, research engineer at the Ecole Polytechnique who dedicated her thesis to ADASOPS, Xavier Solinas, CNRS research engineer in electronics, Adeline Bonvalet, CNRS research engineer, and Manuel Joffre, CNRS research director and professor at the Ecole Polytechnique.
The other experimental approach used, CPU (Chirped Pulse Upconversion), which has been developed for a longer time in the laboratory, has made it possible to detect the infrared spectrum with a spectral resolution that is much higher than that of conventional infrared detector arrays.
The FAP enzyme experiments were adapted to the particular sensitivity of the enzyme, which must be renewed for each absorbed pulse at a rate of 1 kHz. "It was not possible to 'hit' the sample several times as we usually do," explains Marten Vos, "and the tandem work of Adeline and Damien was essential to find the right experimental conditions." The experiments also benefited from the help of Assia Benachir, an intern from the Bachelor program in the laboratory, particularly during the preparation of the measurement campaign. The researchers were also helped by Bo Zhuang, PhD student at LOB, who worked on the data analysis.
In summary, the work carried out at the LOB allowed to follow the formation of the CO2 product in 300 ps, to establish its unexpected partial transformation in 100 ns, and to follow its release towards the protein environment in 1.5 µs, thanks to a very slight spectral shift.
These kinetic data are complementary to transient structural data obtained by other teams of the consortium by time-resolved crystallography and cryo-trapping. Another contribution of the LOB concerns the work of Alexey Aleksandrov, CNRS researcher, who performed molecular simulations to analyze the dynamics of the CO2 spectrum and to explain the unusual curvature of the flavin in the enzyme.
A team effort within an international consortium
It is the combined interpretation of the results of various experimental and theoretical approaches by the international consortium that gives a detailed picture, at the atomic scale, of the enzyme at work. The multidisciplinary study combined bioengineering work, optical and vibrational spectroscopies, static and kinetic crystallography performed with synchrotrons or an X-ray free electron laser, and quantum chemical calculations.
This study was funded by the French National Research Agency (ANR SNAPsHOTs - 18-CE11-0021, coordinated by Frédéric Beisson) and the contribution of the Laboratory of Optics and Biosciences is a very good example of the synergy between advanced optics and the competence in fundamental biology of its teams.
> Read the publication in Science
> Read the national press release
*LOB: a joint research unit CNRS, École Polytechnique - Institut Polytechnique de Paris, Inserm