E. coli Nickel‐Iron Hydrogenase 1 Catalyses Non‐native Reduction of Flavins: Demonstration for Alkene Hydrogenation by Old Yellow Enzyme Ene‐reductases**

Abstract

A new activity for the [NiFe] uptake hydrogenase 1 of Escherichia coli (Hyd1) is presented. Direct reduction of biological flavin cofactors FMN and FAD is achieved using H 2 as a simple, completely atom-economical reductant. The robust nature of Hyd1 is exploited for flavin reduction across a broad range of temperatures (25-70 8C) and extended reaction times. The utility of this system as a simple, easy to implement FMNH 2 or FADH 2 regenerating system is then demonstrated by supplying reduced flavin to Old Yellow Enzyme "enereductases" to support asymmetric alkene reductions with up to 100 % conversion. Hyd1 turnover frequencies up to 20.4 min À1 and total turnover numbers up to 20 200 were recorded during flavin recycling. Scheme 1. Oxidized (left) and reduced (right) FMN or FAD cofactors.

Boosting the Productivity of H2-Driven Biocatalysis in a Commercial Hydrogenation Flow Reactor Using H2 From Water Electrolysis

“…In our previous batch studies, we focused on co-immobilizing a separate NAD + reductase (a protein possessing just the NAD + reductase activity of the SH) with a more robust hydrogenase (E. coli hydrogenase 1, "Hyd1") onto carbon particles; this system yielded much higher stability and cofactor regeneration activity compared with the SH. For reference, we have demonstrated that Hyd1 itself maintains activity after >5 days of continual H 2driven operation (FMN recycling in this case) at room temperature (Joseph Srinivasan et al, 2021). For this two-enzyme catalyst, the carbon plays dual roles, acting as an enzyme support and also electronically linking the enzymes by conducting electrons generated via H 2 -oxidation by the Hyd1 to the NAD + reductase, thereby powering the NAD + reductase to regenerate NADH.…”

“…Biocatalysts for H 2 -driven cofactor recycling (SH and Hyd1) were prepared in-house. Detailed protocols for expression and purification of the hydrogenases [Escherichia coli hydrogenase 1, Hyd1 (Joseph Srinivasan et al, 2021), and R. eutropha soluble hydrogenase, SH (Lauterbach and Lenz, 2013)] have been described previously. Alcohol dehydrogenase ADH-105 was provided by Johnson Matthey in the His-tagged and purified form.…”

“…It was also found that the SH/C deactivates rapidly at >40°C in the hydrogenation flow reactor (see Supplementary Figure S4, Supporting Information). In contrast Hyd1 has excellent temperature stability (Joseph Srinivasan et al, 2021). To determine the activity lifetime of SH + Hyd1/C at elevated temperatures, three identical biocatalytic cartridges containing SH + Hyd1/C were prepared and were tested under conditions of NAD + (5 mM) in buffer, at a liquid flow rate 0.1 ml min −1 in the flow reactor at 30°C, 45°C or 55°C, respectively.…”

Ene‐Reductase: A Multifaceted Biocatalyst in Organic Synthesis

“…Lately, a report by the Vincent group suggested that Nickel‐Iron Hydrogenase 1 of Escherichia Coli can reduce the flavin cofactor using H 2 as the reductant, [66] with a turnover frequency of up to 20.4 min −1 . The Hydrogenase enzyme could recycle the reduced cofactor using clean atom efficient reductant H 2 that helped to continue the asymmetric reduction by ene‐reductases.…”

“…[64] Recently, a novel affinity tag ((HR) 4 -tag) (a combination of polyHis-tag and polyArg-tag) was used to immobilize ene-reductase enzymes on non-functionalized magnetic nanoparticles. [65] Lately, a report by the Vincent group suggested that Nickel-Iron Hydrogenase 1 of Escherichia Coli can reduce the flavin cofactor using H 2 as the reductant, [66] with a turnover frequency of up to 20.4 min À 1 . The Hydrogenase enzyme could recycle the reduced cofactor using clean atom efficient reductant H 2 that helped to continue the asymmetric reduction by ene-reductases.…”