Biocatalyst–artificial metalloenzyme cascadebased on alcohol dehydrogenase

摘要

Chemo-enzymatic cascades of enzymes with transition metal catalysts can offer efficient syntheticstrategies, but their development is challenging due to the incompatibility between proteins andtransition metal complexes. Rhodium catalysts can be combined with alcohol dehydrogenases toregenerate nicotinamide cofactors using formate as the hydride donor. However, their use is limited, dueto binding of the metals to residues on the enzyme surface, leading to mutual enzyme and catalystinactivation. In this work, we replaced the zinc from Thermoanaerobacter brockii alcohol dehydrogenase(TbADH) with Rh(III) catalysts possessing nitrogen donor ligands, by covalent conjugation to the activesite cysteine, to create artificial metalloenzymes for NADP+ reduction. TbADH was used as proteinscaffold for both alcohol synthesis and the recycling of the cofactor, by combination of the chemicallymodified species with the non-modified recombinant enzyme. Stability studies revealed that theincorporation of the catalysts into the TbADH pocket provided a shielding environment for the metalcatalyst, resulting in increased stability of both the recycling catalyst and the ADH. The reduction ofa representative ketone using this novel alcohol dehydrogenase–artificial formate dehydrogenasecascade yielded better conversions than in the presence of free metal catalyst.