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.

Ion channel gating is essential for cellular homeostasis and is tightly controlled. In some eukaryotic and most bacterial ligand-gated K+ channels, RCK domains regulate ion fluxes. Until now, a single regulatory mechanism has been proposed for all RCK-regulated channels, involving signal transduction from the RCK domain to the gating area. Here, we present an inactive ADP-bound structure of KtrAB from Vibrio alginolyticus, determined by cryo-electron microscopy, which, combined with EPR spectroscopy and molecular dynamics simulations, uncovers a novel regulatory mechanism for ligand-induced action at a distance. Exchange of activating ATP to inactivating ADP triggers short helical segments in the K+-translocating KtrB dimer to organize into two long helices that penetrate deeply into the regulatory RCK domains, thus connecting nucleotide-binding sites and ion gates. As KtrAB and its homolog TrkAH have been implicated as bacterial pathogenicity factors, the discovery of this functionally relevant inactive conformation may advance structure-guided drug development.DOI: http://dx.doi.org/10.7554/eLife.24303.001

Phosphocholine molecules decorating bacterial cell wall teichoic acids and outer-membrane lipopolysaccharide have fundamental roles in adhesion to host cells, immune evasion, and persistence. Bacteria carrying the operon that performs phosphocholine decoration synthesize phosphocholine after uptake of the choline precursor by LicB, a conserved transporter among divergent species. Streptococcus pneumoniae is a prominent pathogen where phosphocholine decoration plays a fundamental role in virulence. Here, we present cryo–electron microscopy and crystal structures of S. pneumoniae LicB, revealing distinct conformational states and describing architectural and mechanistic elements essential to choline import. Together with in vitro and in vivo functional characterization, we found that LicB displays proton-coupled import activity and promiscuous selectivity involved in adaptation to choline deprivation conditions, and describe LicB inhibition by synthetic nanobodies (sybodies). Our results provide previously unknown insights into the molecular mechanism of a key transporter involved in bacterial pathogenesis and establish a basis for inhibition of the phosphocholine modification pathway across bacterial phyla.

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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