Characterization of the Novel Ene Reductase Ppo-Er1 from Paenibacillus Polymyxa

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.

Applications of Ene-Reductases in the Synthesis of Flavors and Fragrances

“…So far, various new ERs have been identified to exhibit the ability to reduce carvone, including KYE 1, 40 Yers-ER, 40 Gox0502 (from Gluconobacter oxidans 621H), 76 Gox2684 (from Gluconobacter oxidans 621H), 76 Dbr2 (from Artemisia annua), 77 PETNR, 38,39 TOYE, 35 LacER (from Lactobacillus casei str. Zhang), 78 SYE-4 (from Shewanella oneidensis), 79 XenA (from Pseudomonas putida ATCC 17453), 80 XenB (from Pseudomonas putida ATCC 17453), 80 NemA (from Pseudomonas putida ATCC 17453), 80 YqiG, 36 OYE2p, 21 Ppo-Er1, 44 and PaER (from Pichia angusta). 42 The introduction of emerging technologies (such as headspace-solid phase microextraction, In situ substrate feeding and product removal and protein modification engineering) often brings new vitality to the biotransformation catalyzed by ERs.…”

Loop 6 and the β‐hairpin flap are structural hotspots that determine cofactor specificity in the FMN‐dependent family of ene‐reductases

“…Ene-reductases (ERs) represent a large group of oxidoreductases that catalyze the stereospecific reduction of carbon-carbon double bonds adjacent to an electron-withdrawing group. Among ERs, flavin mononucleotide (FMN)-dependent enzymes of the Old Yellow Enzyme (OYE) family (EC1.6.99.1) have been studied extensively since the isolation and characterization of the first member from Saccharomyces pastorianus (OYE1) by Warburg and Christian in 1932 [1,2]. The last 90 years have witnessed the discovery of many more homologs in bacteria, fungi, and plants.…”

Development of an Ene Reductase-Based Biocatalytic Process for the Production of Flavor Compounds

“…Building on the availability of a diverse ER wild-type library in our laboratory, , we set out to develop biocatalytic processes for the manufacture of interesting olfactory compounds, among others, decanal. Decanal, also known as aldehyde C-10, has a very pungent, sweet, floral, and waxy orange peel-like odor, and consequently it is used, for example, in the creation of citrus flavors .…”

“…3,17,23 However, access to robust and efficient enzymes possessing a broad substrate scope is a general and crucial prerequisite in the development of biocatalytic processes to be run at industrial scale. 28−30 Building on the availability of a diverse ER wild-type library in our laboratory, 15,31 we set out to develop biocatalytic processes for the manufacture of interesting olfactory compounds, among others, decanal. Decanal, also known as aldehyde C-10, has a very pungent, sweet, floral, and waxy orange peel-like odor, and consequently it is used, for example, in the creation of citrus flavors.…”