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A process using an engineered phenylalanine ammonia lyase (PAL) enzyme was developed as part of an alternative route to a key intermediate of olodanrigan (EMA401). In the first part of this report,...

Eumelanin is a naturally occurring skin pigment which is responsible for developing a suntan. The complex structure of eumelanin consists of π-stacked oligomers of various indole derivatives, such as the monomeric building block 5,6-dihydroxyindole (DHI). In this work, we present an ab initio wave-function study of the absorption behavior of DHI oligomers and of doubly and triply π-stacked species of these oligomers. We have simulated the onset of the electronic absorption spectra by employing the MP2 and the linear-response CC2 methods. Our results demonstrate the effect of an increasing degree of oligomerization of DHI and of an increasing degree of π-stacking of DHI oligomers on the onset of the absorption spectra and on the degree of red-shift toward the visible region of the spectrum. We find that π-stacking of DHI and its oligomers substantially red-shifts the onset of the absorption spectra. Our results also suggest that the optical properties of biological eumelanin cannot be simulated by considering the DHI building blocks alone, but instead the building blocks indole-semiquinone and indole-quinone have to be considered as well. This study contributes to advancing the understanding of the complex photophysics of the eumelanin biopolymer.

Optical control of cellular processes is an emerging approach for studying biological systems, affording control with high spatial and temporal resolution. Specifically designed artificial photoswitches add an interesting extension to naturally occurring light-regulated functionalities. However, despite a great deal of structural information, the generation of new tools cannot be based fully on rational design yet; in many cases design is limited by our understanding of molecular details of light activation and signal transduction. Our biochemical and biophysical studies on the established optogenetic tool PA-Rac1, the photoactivatable small GTPase Rac1, reveal how unexpected details of the sensor-effector interface, such as metal coordination, significantly affect functionally important structural elements of this photoswitch. Together with solution scattering experiments, our results favor differences in the population of pre-existing conformations as the underlying allosteric activation mechanism of PA-Rac1, rather than the assumed release of the Rac1 domain from the caging photoreceptor domain. These results have implications for the design of new optogenetic tools and highlight the importance of including molecular details of the sensor-effector interface, which is however difficult to assess during the initial design of novel artificial photoswitches.

Extensive interest in photosensory proteins stimulated computational studies of flavins and flavoproteins in the past decade. This review is dedicated to the three central topics of these studies: calculations of flavin UV-visible and IR spectra, simulated dynamics of photoreceptor proteins, and flavin photochemistry. Accordingly, this chapter is divided into three parts; each part describes corresponding computational protocols, summarizes computational results, and discusses the emerging mechanistic picture.

The blue light using FAD (BLUF) photosensory protein domain is activated by a unique photoreaction that results in a hydrogen-bond rearrangement around the flavin chromophore. The chemical structure of the hydrogen bond switch is a long-standing debate: The two main hypotheses postulate rotation as opposed to tautomerization of a conserved glutamine residue. Attempts to resolve the debate were inconclusive so far, despite numerous experimental and computational studies. Here we propose physical criteria for the dark and light state structures as well as for the light-activation process to evaluate existing models of BLUF using quantum-chemical calculations. The glutamine rotamer assignment of the crystal structure with the pdb code 1YRX does not satisfy our criteria because after equilibrating the intermolecular forces the glutamine rotamer in 1YRX is incompatible with the experimental density. We identified the root of the mechanistic controversy in the incorrect glutamine rotamer assignment of 1YRX . Furthermore, we show that the glutamine side chain may rotate without light activation in the BLUF dark state. Finally, we demonstrate that the tautomerized glutamine is consistent with our criteria and observations of the BLUF light state.

Many extremophilic organisms require unusual enzymes to help them survive in harsh environments. For example, acid-loving hyperthermophilic Archaea found in the bubbling mud of volcanic solfataras are able to oxidize reduced sulphur compounds. The X-ray crystal structure of a carbon disulphide (CS2) hydrolase from an Acidianus strain isolated from the Solfatara volcano near Naples, Italy, has now been determined. The enzyme, which converts CS2 into hydrogen sulphide and carbon dioxide, has a typical carbonic anhydrase fold and active site, although CO2 is not a substrate for the enzyme. This suggests that CS2 hydrolase is an example of divergent evolution, where a new enzyme has emerged through the evolution of a new quaternary structure rather than through mutations of the active site. Extremophilic organisms require specialized enzymes for their exotic metabolisms. Acid-loving thermophilic Archaea that live in the mudpots of volcanic solfataras obtain their energy from reduced sulphur compounds such as hydrogen sulphide (H2S) and carbon disulphide (CS2)1,2. The oxidation of these compounds into sulphuric acid creates the extremely acidic environment that characterizes solfataras. The hyperthermophilic Acidianus strain A1-3, which was isolated from the fumarolic, ancient sauna building at the Solfatara volcano (Naples, Italy), was shown to rapidly convert CS2 into H2S and carbon dioxide (CO2), but nothing has been known about the modes of action and the evolution of the enzyme(s) involved. Here we describe the structure, the proposed mechanism and evolution of a CS2 hydrolase from Acidianus A1-3. The enzyme monomer displays a typical β-carbonic anhydrase fold and active site, yet CO2 is not one of its substrates. Owing to large carboxy- and amino-terminal arms, an unusual hexadecameric catenane oligomer has evolved. This structure results in the blocking of the entrance to the active site that is found in canonical β-carbonic anhydrases and the formation of a single 15-A-long, highly hydrophobic tunnel that functions as a specificity filter. The tunnel determines the enzyme’s substrate specificity for CS2, which is hydrophobic. The transposon sequences that surround the gene encoding this CS2 hydrolase point to horizontal gene transfer as a mechanism for its acquisition during evolution. Our results show how the ancient β-carbonic anhydrase, which is central to global carbon metabolism, was transformed by divergent evolution into a crucial enzyme in CS2 metabolism.

Photoinduced electron transfer from tyrosine to the flavin chromophore is involved in activation of BLUF (sensor of blue light using FAD) photoreceptors. We studied the electron transfer (ET) coupled with proton-transfer (PT) reactions, by means of XMCQDPT2//CASSCF calculations on a molecular cluster model. By defining a minimum active space in the CASSCF calculations, we could compute the entire photoreaction pathway. We find that the crossing of the locally excited and ET states is located along the flavin bond-stretching coordinate. The ET state is stabilized by a proton transfer from the electron donor to the electron acceptor. We mapped two different PT pathways from tyrosine to flavin via the conserved glutamine. These reactions generate a tautomeric form of glutamine. Along the PT coordinates, we find geometries where the ET and the electronic ground states degenerate. At the state crossing structures, either formation of the ground state biradical intermediate or a relaxation back to the Franck-Condon minimum takes places. The computed relaxation pathways reveal that the hydrogen bonds involving glutamine in the chromophore-binding pocket control BLUF photoefficiency.