Loop Swapping as a Potent Approach to Increase Ene Reductase Activity with Nicotinamide Adenine Dinucleotide (NADH)

Abstract

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.

Asymmetric Whole-Cell Bio-Reductions of (R)-Carvone Using Optimized Ene Reductases

“…Four different NostocER1 variants were applied, i.e., the wild type, a loop 1,2a swapped mutant with a highly increased activity with NADH, a loop 1,5 swapped mutant with a highly increased affinity toward NADH, and the combination of these two, a loop 1,5,2a swapped mutant with an increased activity and affinity with NADH. Details of differences in the primary structure and kinetic parameters can be found in a previous work [12] and Table 1.…”

“…Therefore, part of the harvested E. coli cells were mechanically disrupted and the enzymatic activities were determined in the supernatant. Since the four applied NostocER1-variants possess different activities with NADH [12], a direct comparison of the specific activities is not possible. Therefore, the determined activities were normalized to the specific activity using 500 µM NADH (k 500 ) of the respective purified NostocER1-variant (Table 1).…”

“…However, further improved biocatalysts were generated by exchanging the NostocER1 wild type with different NADH-accepting NostocER1-variants (Table 2). The implementation of the Loop 1,2a NostocER1-variant, which possesses a highly increased catalytic constant with NADH [12], resulted in significant improvement. Thereby, a yield of 95.7% was achieved after a process time of 5 h. The initial product formation rate was increased 2.1-fold to 2.8 ± 0.1 mmol h −1 g CDW −1 and the space-time yield after ≥96% conversion was increased 1.8-fold to 57.4 ± 0.1 mmol L −1 h −1 when compared to literature [19].…”

“…Furthermore, a high and stereospecific conversion of 96.8% of the substrate was reached within 9 h of processing time [19]. In this study, this biocatalyst was further improved by applying NADH-accepting variants of NostocER1 [12], which results in up to 99.4% conversion of 300 mM ( R )-carvone within 6 h of process time. The product formation rate was increased by a factor of up to 2.1 and the space-time yield by a factor of 1.8.…”

“…In a previous work, we were able to influence the cosubstrate preference of the cyanobacterial ene reductase 1 from Nostoc sp. PCC7120 (NostocER1) [12]. This was achieved by swapping loop regions that participate in cosubstrate binding.…”