Abstract:Asymmetric hydrogenation of activated alkenes catalysed by ene-reductases from the old yellow enzyme family (OYEs) leading to chiral products is of potential interest for industrial processes. OYEs' dependency on the pyridine nucleotide coenzyme can be circumvented through established artificial hydride donors such as nicotinamide coenzyme biomimetics (NCBs). Several OYEs were found to exhibit higher reduction rates with NCBs. In this review, we describe a new classification of OYEs into three main classes by … Show more
“…The predicted amino acid sequences were subsequently designated as Rhr ER 301, Rhr ER 2718 and Rhr ER 5439. A multiple sequence alignment was performed with 2–3 sequences from each class of known ene-reductases (Scholtissek et al 2017) using the online available Clustal Omega alignment tool (Sievers et al 2011). As shown in the supplemental information (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…It is noteworthy that these 2 enzymes seem to have an extended loop between α-helix 6 and β-sheet 6, which could all point towards a different substrate specificity of these enzymes.Fig. 2Phylogenetic relationship of Rhr ERs to other OYEs with known function from different classes (Scholtissek et al 2017). The tree was constructed using the “One-Click” Mode on Phylogeny.fr (Dereeper et al 2008), using the same OYEs as in the sequence alignment (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Phylogenetic relationship of Rhr ERs to other OYEs with known function from different classes (Scholtissek et al 2017). The tree was constructed using the “One-Click” Mode on Phylogeny.fr (Dereeper et al 2008), using the same OYEs as in the sequence alignment (Fig.…”
Rhodococcus strains are ubiquitous in nature and known to metabolise a wide variety of compounds. At the same time, asymmetric reduction of C=C bonds is important in the production of high-valued chiral building blocks. In order to evaluate if Rhodococci can be used for this task, we have probed several Rhodococcus rhodochrous and R. erythropolis strains for ene-reductase activity. A series of substrates including activated ketones, an aldehyde, an imide and nitro-compound were screened using whole cells of seven Rhodococcus strains. This revealed that whole cells of all Rhodococcus strains showed apparent (S)-selectivity towards ketoisophorone, while most other organisms show (R)-selectivity for this compound. Three putative ene-reductases from R. rhodochrous ATCC 17895 were heterologously expressed in Escherichia coli. One protein was purified and its biocatalytic and biochemical properties were characterised, showing typical (enantioselective) properties for class 3 ene-reductases of the old yellow enzyme family.Electronic supplementary materialThe online version of this article (10.1007/s00253-018-8984-7) contains supplementary material, which is available to authorized users.
“…The predicted amino acid sequences were subsequently designated as Rhr ER 301, Rhr ER 2718 and Rhr ER 5439. A multiple sequence alignment was performed with 2–3 sequences from each class of known ene-reductases (Scholtissek et al 2017) using the online available Clustal Omega alignment tool (Sievers et al 2011). As shown in the supplemental information (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…It is noteworthy that these 2 enzymes seem to have an extended loop between α-helix 6 and β-sheet 6, which could all point towards a different substrate specificity of these enzymes.Fig. 2Phylogenetic relationship of Rhr ERs to other OYEs with known function from different classes (Scholtissek et al 2017). The tree was constructed using the “One-Click” Mode on Phylogeny.fr (Dereeper et al 2008), using the same OYEs as in the sequence alignment (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Phylogenetic relationship of Rhr ERs to other OYEs with known function from different classes (Scholtissek et al 2017). The tree was constructed using the “One-Click” Mode on Phylogeny.fr (Dereeper et al 2008), using the same OYEs as in the sequence alignment (Fig.…”
Rhodococcus strains are ubiquitous in nature and known to metabolise a wide variety of compounds. At the same time, asymmetric reduction of C=C bonds is important in the production of high-valued chiral building blocks. In order to evaluate if Rhodococci can be used for this task, we have probed several Rhodococcus rhodochrous and R. erythropolis strains for ene-reductase activity. A series of substrates including activated ketones, an aldehyde, an imide and nitro-compound were screened using whole cells of seven Rhodococcus strains. This revealed that whole cells of all Rhodococcus strains showed apparent (S)-selectivity towards ketoisophorone, while most other organisms show (R)-selectivity for this compound. Three putative ene-reductases from R. rhodochrous ATCC 17895 were heterologously expressed in Escherichia coli. One protein was purified and its biocatalytic and biochemical properties were characterised, showing typical (enantioselective) properties for class 3 ene-reductases of the old yellow enzyme family.Electronic supplementary materialThe online version of this article (10.1007/s00253-018-8984-7) contains supplementary material, which is available to authorized users.
“…ERs are a class of redox enzymes that catalyze the asymmetric reduction of activated C=C bonds to form stereogenic centers . Their ability to catalyze reductions and their wide acceptance of different substrates are driving the exploitation of ERs for novel applications in redox biocatalysis and their implementation in industrial processes . The ER catalytic reaction resembles an asymmetric Michael‐type addition of a chiral hydride to an enone.…”
Nicotinamide and pyridine‐containing conjugates have attracted a lot of attention in research as they have found use in a wide range of applications including as redox flow batteries and calcium channel blockers, in biocatalysis, and in metabolism. The interesting redox character of the compounds’ pyridine/dihydropyridine system allows them to possess very similar characteristics to the natural chiral redox agents NAD+/NADH, even mimicking their functions. There has been considerable interest in designing and synthesizing NAD+/NADH mimetics with similar redox properties. In this research, three nicotinamide conjugates were designed, synthesized, and characterized. Molecular structures obtained through X‐ray crystallography were obtained for two of the conjugates, thereby providing more detail on the bonding and structure of the compounds. The compounds were then further evaluated for biochemical properties, and it was found that one of the conjugates possessed similar functions and characteristics to the natural NADH. This compound was evaluated in the active enzyme, enoate reductase; like NADH, it was shown to help reduce the C=C double bond of three substrates and even outperformed the natural coenzyme. Kinetic data are reported.
“…[7] During the oxidative half reaction this hydride reduces the electron deficient double bond in an anti-specific manner creating up to two stereogenic centers. [6] All currently known members of the OYE family have essentially the same tertiary structure, an (βα) 8 -barrel structure or so-called triose phosphate isomerase (TIM)-barrel fold. [4] This means that, eight alternating α-helices and parallel β-sheets form a barrel-like structure, in which the sheets shape the inner wall and the helices the outer wall of the barrel.…”
The asymmetric reduction of alkenes is a widely used transformation in industry. Ene reductases (ERs) are (βα) 8 -barrel folded enzymes capable of catalyzing this hydrogenation reaction. At the expense of nicotinamide coenzymes, ERs can reduce a wide range of electron-deficient alkenes in an anti-specific manner and with high regio-and stereoselectivities. However, a cost-effective industrial use of these enzymes is hampered, since most ERs prefer nicotinamide adenine dinucleotide phosphate (NADPH) to the more stable and less expensive non-phosphorylated nicotinamide adenine dinucleotide (NADH) as coenzyme. Here, we demonstrate an approach to both modify the biocatalysts coenzyme selectivity and strongly increase the activity and affinity with NADH. By swapping loop regions of the cyanobacterial NostocER1 for the corresponding regions of two NADH-favoring ERs, a strong alteration of the biocatalyst's coenzyme binding was achieved. This made possible a transfer of the respective donor-ER kinetic parameters to NostocER1. Additionally, outperformance of both donors in terms of activity was achieved through combinatorial swapping of loops of both species. These findings demonstrate the high potential of loop swapping as protein engineering approach to selectively optimize the coenzyme binding of ERs.
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