期刊
CHEMBIOCHEM
卷 -, 期 -, 页码 -出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.202300409
关键词
biocatalysis; cofactor regeneration; ferredoxin; flavin adenine dinucleotide; oxidoreductases; reduction
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.
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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