Novel Old Yellow Enzyme Subclasses

Abstract

Many drug candidate molecules contain at least one chiral centre, and consequently, the development of biocatalytic strategies to complement existing metal‐ and organocatalytic approaches is of high interest. However, time is a critical factor in chemical process development, and thus, the introduction of biocatalytic steps, even if more suitable, is often prevented by the limited availability of off‐the‐shelf enzyme libraries. To expand the biocatalytic toolbox with additional ene reductases, we screened 19 bacterial strains for double bond reduction activity by using the model substrates cyclohexanone and carvone. Overall, we identified 47 genes coding for putative ene reductases. Remarkably, bioinformatic analysis of all genes and the biochemical characterization of four representative novel ene reductases led us to propose the existence of two new Old Yellow Enzyme subclasses, which we named OYE class III and class IV. Our results demonstrate that although, on a DNA level, each new OYE subclass features a distinct combination of sequence motifs previously known from the classical and the thermophilic‐like group, their substrate scope more closely resembles the latter subclass.

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

“…The enzyme Ppo-Er1 from P. polymyxa was discovered during the screening of 19 bacterial wild-type strains from the Culture Collection of Switzerland, as previously described [15]. Ppo-Er1 (41.3 kDa) is characterized by a substantial sequence similarity with the old yellow enzyme YqiG from Bacillus subtilis (50%) [34], Bac-OYE2 from Bacillus sp.…”

“…Notably, amino acid sequence alignments of OYE homologs show high conservation in specific regions of the proteins, such as residues involved in catalysis, FMN, and substrate binding [7,15,23]. To account for these differences in sequence and the resulting structural features, the old yellow enzyme family can be further divided into five subclasses [15].…”

“…Notably, amino acid sequence alignments of OYE homologs show high conservation in specific regions of the proteins, such as residues involved in catalysis, FMN, and substrate binding [7,15,23]. To account for these differences in sequence and the resulting structural features, the old yellow enzyme family can be further divided into five subclasses [15]. While enzyme members of the subclass I, also termed "classical" old yellow enzymes, and class II, introduced by Scrutton's group in 2010 and dubbed "thermophilic-like" [23], have been well explored [7,14], the recently described class III-V are less well investigated [15,24].…”

“…Synthetic applications of ene reductases are manifold and range from the preparation of profens [25][26][27] and chiralγ-amino acids [28][29][30] to the synthesis of chiral phosphonates [31] and nitroalkanes [32], precursors in the synthesis of pharmaceutically active ingredients. To further promote an off-the-shelve synthetic use of ene reductases, which can reduce the time and cost of the implementation of a biocatalytic step into a process significantly, we set out to expand the available biocatalytic toolbox [15]. In this context, not only the discovery and engineering of novel ene reductases is of great utility [33], but also a careful characterization of the new biocatalysts is needed as it may lead to the construction of a more targeted enzyme library associated with reduced screening time and costs.…”

“…While enoate reductases, medium-and short-chain dehydrogenases/reductases (MDR and SDR), as well as the recently discovered quinone Figure 1. Overview of the classification within the ene reductase family [15]. QnoR (NADPHdependent quinone reductase like ene-reductases), EnoR (enoate reductase), OYE (old yellow enzyme), MDR (medium-chain dehydrogenase/reductase), and SDR (short-chain dehydrogenase/reductase); Class I (classical OYE); Class II (thermophilic-like OYE) and Class V (fungal OYE).…”