Tamara Reiter

Nicotinamide-independent asymmetric bioreduction of CC-bonds via disproportionation of enones catalyzed by enoate reductases

The asymmetric bioreduction of activated CC-bonds catalyzed by a single flavoprotein was achieved via direct hydrogen transfer from a sacrificial 2-enone or 1,4-dione as hydrogen donor without requirement of a nicotinamide cofactor. Due to its simplicity, this system has clear advantages over conventional FAD-recycling systems.

Regioselective para-Carboxylation of Catechols with a Prenylated Flavin Dependent Decarboxylase

Abstract 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.

Sequential Enzymatic Conversion of α-Angelica Lactone to γ-Valerolactone through Hydride-Independent C=C Bond Isomerization

Abstract A case of hydride‐independent reaction catalyzed by flavin‐dependent ene‐reductases from the Old Yellow Enzyme (OYE) family was identified. α‐Angelica lactone was isomerized to the conjugated β‐isomer in a nicotinamide‐free and hydride‐independent process. The catalytic cycle of C=C bond isomerization appears to be flavin‐independent and to rely solely on a deprotonation–reprotonation sequence through acid–base catalysis. Key residues in the enzyme active site were mutated and provided insight on important mechanistic features. The isomerization of α‐angelica lactone by OYE2 in aqueous buffer furnished 6.3 mm β‐isomer in 15 min at 30 °C. In presence of nicotinamide adenine dinucleotide (NADH), the latter could be further reduced to γ‐valerolactone. This enzymatic tool was successfully applied on semi‐preparative scale and constitutes a sustainable process for the valorization of platform chemicals from renewable resources.

Biocatalytic Racemization Employing TeSADH: Substrate Scope and Organic Solvent Compatibility for Dynamic Kinetic Resolution

Racemization in combination with a kinetic resolution is the base for a dynamic kinetic resolution (DKR). Biocatalytic racemization was successfully performed for a broad scope of sec‐alcohols by employing a single alcohol dehydrogenase (ADH) variant from Thermoanaerobacter pseudoethanolicus (formerly T. ethanolicus; TeSADH W110A I86A C295A). The catalyst employed as a lyophilized whole cell preparation or cell free extract, which tolerated various non‐water miscible organic solvents under micro‐aqueous or two‐phase conditions, whereby cyclohexane and n‐hexane suited best. Various concepts for combining the enzymatic racemization with an enzymatic kinetic resolution to achieve overall a bis‐enzymatic DKR were evaluated. A proof of concept showed a successful DKR with racemization in aqueous phase combined with acylation in the organic phase.