Abstract
Chemo-enzymatic cascades of enzymes with transition metal catalysts can offer efficient synthetic strategies, but their development is challenging due to the incompatibility between proteins and transition metal complexes. Rhodium catalysts can be combined with alcohol dehydrogenases to regenerate nicotinamide cofactors using formate as the hydride donor. However, their use is limited, due to binding of the metals to residues on the enzyme surface, leading to mutual enzyme and catalyst inactivation. 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 active site cysteine, to create artificial metalloenzymes for NADP+ reduction. TbADH was used as protein scaffold for both alcohol synthesis and the recycling of the cofactor, by combination of the chemically modified species with the non-modified recombinant enzyme. Stability studies revealed that the incorporation of the catalysts into the TbADH pocket provided a shielding environment for the metal catalyst, resulting in increased stability of both the recycling catalyst and the ADH. The reduction of a representative ketone using this novel alcohol dehydrogenase-artificial formate dehydrogenase cascade yielded better conversions than in the presence of free metal catalyst.
Highlights
Alcohol dehydrogenases (ADHs) are valuable biocatalytic tools for the conversion of ketones into chiral alcohols, providing a sustainable and efficient alternative to chiral catalysts based on transition metals.[1]
We replaced the zinc from Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) with Rh(III) catalysts possessing nitrogen donor ligands, by covalent conjugation to the active site cysteine, to create artificial metalloenzymes for NADP+ reduction
TbADH is a Zn-dependent bacterial ADH with a homotetrameric structure, in which the catalytic Zn is bound by residues C37, H59 and D150.36 Its distinguishing feature compared to other medium-chain Zn-dependent ADHs is the absence of a second, structural Zn-binding site
Summary
Alcohol dehydrogenases (ADHs) are valuable biocatalytic tools for the conversion of ketones into chiral alcohols, providing a sustainable and efficient alternative to chiral catalysts based on transition metals.[1]. The reduction of NAD(P)+ has been achieved with metal catalysts incorporated into proteins, either by covalent linking to papain,[28] or by supramolecular insertion using the wellestablished biotin–streptavidin system.[29] In this context, Ward and co-workers reported that the protein host provided a shielding environment for the metal catalyst against inhibitory species present in solution This concept of “molecular compartmentalisation” has been demonstrated by an increased activity of the protein-bound compared to the free metal complex, when used in situ with various enzymes dependent on NAD(P)H or on NAD(P)H mimics.[30,31,32] Whilst a range of cofactordependent biocatalysts have been employed in conjunction with arti cial metalloenzymes, such as ene reductases, glucose dehydrogenase and 2-hydroxybiphenyl monooxygenase, a cascade reaction with alcohol dehydrogenases remains to be developed. The potential of the protein to prevent metal inactivation will be studied and the new arti cial metalloenzymes will be compared with nonenzymatic cofactor regeneration catalysts in a synthetic cascade
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