An Enzymatic Cofactor Regeneration System for the in-Vitro Reduction of Isolated C=C Bonds by Geranylgeranyl Reductases
CHEMBIOCHEM(2024)
摘要
Cofactor regeneration systems are of major importance for the applicability of oxidoreductases in biocatalysis. Previously, geranylgeranyl reductases have been investigated for the enzymatic reduction of isolated C=C bonds. However, an enzymatic cofactor-regeneration system for in vitro use is lacking. In this work, we report a ferredoxin from the archaea Archaeoglobus fulgidus that regenerates the flavin of the corresponding geranylgeranyl reductase. The proteins were heterologously produced, and the regeneration was coupled to a ferredoxin reductase from Escherichia coli and a glucose dehydrogenase from Bacillus subtilis, thereby enabling the reduction of isolated C=C bonds by purified enzymes. The system was applied in crude, cell-free extracts and gave conversions comparable to those of a previous method using sodium dithionite for cofactor regeneration. Hence, an enzymatic approach to the reduction of isolated C=C bonds can be coupled with common systems for the regeneration of nicotinamide cofactors, thereby opening new perspectives for the application of geranylgeranyl reductases in biocatalysis. A ferredoxin from Archaeoglobus fulgidus has been identified that regenerates the cofactor in the corresponding geranylgeranyl reductase (GGR) for the reduction of isolated C=C bonds. The reaction was coupled to an unspecific ferredoxin reductase from E. coli and a glucose dehydrogenase/glucose regeneration system for NAD(P)+ to enable reactions with purified enzymes. Moreover, reactions can be performed in E. coli cell-free extracts.image
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关键词
biocatalysis,cofactor regeneration,ferredoxin,flavin adenine dinucleotide,oxidoreductases,reduction
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