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.
<p>Robust
[NiFe] hydrogenase 1 (Hyd1) from <i>Escherichia
coli</i> is shown to have non-native, H<sub>2</sub>-dependent activity for FMN
and FAD reduction, and to function as a promising recycling system for FMNH<sub>2</sub>
supply to flavoenzymes for chemical synthesis, giving a total turnover number
over 10 thousand when coupled with an Old Yellow Enzyme ene reductase. </p>
Hydrogenase-mediated reduction of flavin mononucleotide by H2 is exploited to enable cleaner application of nitroreductase enzymes for reduction of aromatic nitro functional groups. This turns the overall reaction into a biocatalytic hydrogenation. Use of flavin-containing nitroreductases in industrial biotechnology typically relies upon NADH or NADPH as reductant, together with glucose dehydrogenase and glucose as a regeneration system for the reduced nicotinamide cofactor, with 3 equivalents of the carbon-intensive glucose required for a single 6-electron nitro to amine conversion. We show here that reduced flavin mononucleotide is an alternative reductant for nitroreductases, and by combining this with H2-driven recycling of reduced flavin, we avoid glucose, thereby enabling atom-efficient biocatalytic nitro reductions. We compare this biocatalytic system, via green chemistry metrics, to existing strategies for biocatalytic nitro-group reductions, particularly with respect to replacing glucose with H2 gas. We take steps towards demonstrating industrial viability: we report an overexpression system for E. coli hydrogenase 1, giving a 12-fold improvement in enzyme yield; we show a reaction in which the hydrogenase exhibits > 26,000 enzyme turnovers; and we demonstrate reasonable solvent tolerance of the hydrogenase and flavin reduction system which would enable reaction intensification.
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.
<p>Robust
[NiFe] hydrogenase 1 (Hyd1) from <i>Escherichia
coli</i> is shown to have non-native, H<sub>2</sub>-dependent activity for FMN
and FAD reduction, and to function as a promising recycling system for FMNH<sub>2</sub>
supply to flavoenzymes for chemical synthesis, giving a total turnover number
over 10 million when coupled with an Old Yellow Enzyme ene reductase. </p>
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