The utilization of CO2 as a carbon source for organic synthesis meets the urgent demand for more sustainability in the production of chemicals. Herein, we report on the enzyme‐catalyzed para‐carboxylation of catechols, employing 3,4‐dihydroxybenzoic acid decarboxylases (AroY) that belong to the UbiD enzyme family. Crystal structures and accompanying solution data confirmed that AroY utilizes the recently discovered prenylated FMN (prFMN) cofactor, and requires oxidative maturation to form the catalytically competent prFMNiminium species. This study reports on the in vitro reconstitution and activation of a prFMN‐dependent enzyme that is capable of directly carboxylating aromatic catechol substrates under ambient conditions. A reaction mechanism for the reversible decarboxylation involving an intermediate with a single covalent bond between a quinoid adduct and cofactor is proposed, which is distinct from the mechanism of prFMN‐associated 1,3‐dipolar cycloadditions in related enzymes.

Stressosomes are stress-sensing protein complexes widely conserved among bacteria. Although a role in the regulation of the general stress response is well documented in Gram-positive bacteria, the activating signals are still unclear, and little is known about the physiological function of stressosomes in the Gram-negative bacteria. Here we investigated the stressosome of the Gram-negative marine pathogen Vibrio vulnificus. We demonstrate that it senses oxygen and identified its role in modulating iron-metabolism. We determined a cryo-electron microscopy structure of the VvRsbR:VvRsbS stressosome complex, the first solved from a Gram-negative bacterium. The structure points to a variation in the VvRsbR and VvRsbS stoichiometry and a symmetry breach in the oxygen sensing domain of VvRsbR, suggesting how signal-sensing elicits a stress response. The findings provide a link between ligand-dependent signaling and an output – regulation of iron metabolism - for a stressosome complex.

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1−GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.

Carboxylierungen unter Verwendung von CO 2 als C 1 -Baustein finden großen Anklang in der nachhaltigen Produktion von Chemikalien.[1] Bis heute werden allerdings nur wenige CO 2 -Fixierungsverfahren im industriellen Maßstab durchgeführt, da ein beträchtlicher Energieaufwand fürd ie Aktivierung der Substrate bençtigt wird. Die Entwicklung und Erforschung von Biokatalysatoren, [2] die unter milden Bedingungen in wässrigem Medium eingesetzt werden kçnnen, erlangten innerhalb der letzten Jahre große Bedeutung als attraktive Alternative zu chemischen Prozessen. [1,3] Während die biokatalytische Carboxylierung von Aldehyden (TPPabhängige Pyruvat-Decarboxylasen), [4] Epoxiden (EpoxidCarboxylase aus Xanthobacter sp.) [5] und Heteroaromaten wie Pyrrolen (Pyrrol-2-carboxylat-Decarboxylase aus Bacillus megaterium) [6] und Indolen (Indol-3-carboxylat-Decarboxylase aus Arthrobacter nicotianae)[2e] auf die natürlichen Substrate beschränkt ist, wurden vielversprechende Ergebnisse in der Biocarboxylierung von Phenolen und Styrolen erzielt. ortho-Benzoesäure-Decarboxylasen und Phenolsäu-re-Decarboxylasen weisen eine weniger strikte Substratspezifitäta uf und wurden unter Beibehaltung ihrer exzellenten Regioselektivitätz ur ortho-b zw. b-Carboxylierung von verschiedenen Phenolen [7] und Styrolen [8] eingesetzt. Anfangs wurde nach geeigneten Enzymen gesucht, um das Repertoire an Biokatalysatoren speziell fürd ie regioselektive para-Carboxylierung von Phenolen zu erweitern. Viele der bereits beschriebenen Enzyme bençtigen entweder eine der Carboxylierung vorgelagerte ATP-abhängige Aktivierung (Phosphorylierung) der Substrate (PhenylphosphatCarboxylasen) [9] oder zeigen, speziell als Reinenzym, einen rapiden Aktivitätsverlust unter aeroben Bedingungen (4-Hydroxybenzoat- [10] und 3,4-Dihydroxybenzoat-Decarboxylasen [2d,11] ). All das und die Tatsache,d ass viele Enzyme ein-

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