Abstract
Ene reductases enable the asymmetric hydrogenation of activated alkenes allowing the manufacture of valuable chiral products. The enzymes complement existing metal- and organocatalytic approaches for the stereoselective reduction of activated C=C double bonds, and efforts to expand the biocatalytic toolbox with additional ene reductases are of high academic and industrial interest. Here, we present the characterization of a novel ene reductase from Paenibacillus polymyxa, named Ppo-Er1, belonging to the recently identified subgroup III of the old yellow enzyme family. The determination of substrate scope, solvent stability, temperature, and pH range of Ppo-Er1 is one of the first examples of a detailed biophysical characterization of a subgroup III enzyme. Notably, Ppo-Er1 possesses a wide temperature optimum (Topt: 20–45 °C) and retains high conversion rates of at least 70% even at 10 °C reaction temperature making it an interesting biocatalyst for the conversion of temperature-labile substrates. When assaying a set of different organic solvents to determine Ppo-Er1′s solvent tolerance, the ene reductase exhibited good performance in up to 40% cyclohexane as well as 20 vol% DMSO and ethanol. In summary, Ppo-Er1 exhibited activity for thirteen out of the nineteen investigated compounds, for ten of which Michaelis–Menten kinetics could be determined. The enzyme exhibited the highest specificity constant for maleimide with a kcat/KM value of 287 mM−1 s−1. In addition, Ppo-Er1 proved to be highly enantioselective for selected substrates with measured enantiomeric excess values of 92% or higher for 2-methyl-2-cyclohexenone, citral, and carvone.
Highlights
Many bioactive molecules contain at least one chiral center rendering the development of effective asymmetric synthesis methods essential for the chemical industry
Ppo-Er1 contains a specific combination of motifs known from the classical and thermophilic-like groups that has been found to be characteristic for class III enzymes [15]: Gln104 and Arg228 predicted to interact with the pyrimidine ring of FMN [22], His 171, and Asn 175 proposed to interact with N1 and N3 of FMN [22,37]; Thr30 suggested to interact with isoalloxazine ring O4 of FMN [38]; and Met29, Leu324, and Arg321, which presumably interact with the dimethyl benzene moiety of FMN
The ready-to-use plasmid consisting of pET-28b(+) vector and the Ppo-Er1 sequence was assembled by Twist Bioscience and a C-terminal His6 tag for protein purification by affinity chromatography was included
Summary
Many bioactive molecules contain at least one chiral center rendering the development of effective asymmetric synthesis methods essential for the chemical industry. Besides the well-established metal- and organocatalytic approaches [1], biocatalytic strategies offer an interesting alternative to install chirality into small molecules. Looking forward, the increasing power of genomic mining and enzyme engineering will allow industrial access to even more enzyme families leading to an expansion of the available biocatalytic toolbox [5]. The families of enzymes collectively known as ene reductases (ERs) catalyze the stereoselective trans- and, more rarely, cis-hydrogenation of activated alkenes [6,7,8,9]. Ene reductases offer a valuable access route to asymmetric compounds, which is complementary to the chemical cis-hydrogenation catalyzed by chiral rhodium or ruthenium phosphine catalysts [10,11]. Medium- and short-chain dehydrogenases/reductases (MDR and SDR), as well as the recently discovered quinone
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