Print Email Facebook Twitter A Tailor-Made Deazaflavin-Mediated Recycling System for Artificial Nicotinamide Cofactor Biomimetics Title A Tailor-Made Deazaflavin-Mediated Recycling System for Artificial Nicotinamide Cofactor Biomimetics Author Drenth, Jeroen (Rijksuniversiteit Groningen) Yang, Guang (Rijksuniversiteit Groningen) Paul, C.E. (TU Delft BT/Biocatalysis) Fraaije, Marco W. (University Medical Center Groningen; Rijksuniversiteit Groningen) Date 2021 Abstract Nicotinamide adenine dinucleotide (NAD) and its 2′-phosphorylated form NADP are crucial cofactors for a large array of biocatalytically important redox enzymes. Their high cost and relatively poor stability, however, make them less attractive electron mediators for industrial processes. Nicotinamide cofactor biomimetics (NCBs) are easily synthesized, are inexpensive, and are also generally more stable than their natural counterparts. A bottleneck for the application of these artificial hydride carriers is the lack of efficient cofactor recycling methods. Therefore, we engineered the thermostable F420:NADPH oxidoreductase from Thermobifida fusca (Tfu-FNO), by structure-inspired site-directed mutagenesis, to accommodate the unnatural N1 substituents of eight NCBs. The extraordinarily low redox potential of the natural cofactor F420H2 was then exploited to reduce these NCBs. Wild-type enzyme had detectable activity toward all selected NCBs, with Km values in the millimolar range and kcat values ranging from 0.09 to 1.4 min-1. Saturation mutagenesis at positions Gly-29 and Pro-89 resulted in mutants with up to 139 times higher catalytic efficiencies. Mutant G29W showed a kcat value of 4.2 s-1 toward 1-benzyl-3-acetylpyridine (BAP+), which is similar to the kcat value for the natural substrate NADP+. The best Tfu-FNO variants for a specific NCB were then used for the recycling of catalytic amounts of these nicotinamides in conversion experiments with the thermostable ene-reductase from Thermus scotoductus (TsOYE). We were able to fully convert 10 mM ketoisophorone with BAP+ within 16 h, using F420 or its artificial biomimetic FOP (FO-2′-phosphate) as an efficient electron mediator and glucose-6-phosphate as an electron donor. The generated toolbox of thermostable and NCB-dependent Tfu-FNO variants offers powerful cofactor regeneration biocatalysts for the reduction of several artificial nicotinamide biomimetics at both ambient and high temperatures. In fact, to our knowledge, this enzymatic method seems to be the best-performing NCB-recycling system for BNAH and BAPH thus far. Subject artificial cofactorsdeazaflavinsenzymatic recycling systemenzyme engineeringFnicotinamide biomimeticsredox enzymology To reference this document use: http://resolver.tudelft.nl/uuid:513e4f13-1627-455a-89fa-0e53e0133892 DOI https://doi.org/10.1021/acscatal.1c03033 ISSN 2155-5435 Source ACS Catalysis, 11 (18), 11561-11569 Part of collection Institutional Repository Document type journal article Rights © 2021 Jeroen Drenth, Guang Yang, C.E. Paul, Marco W. Fraaije Files PDF acscatal.1c03033.pdf 1.49 MB Close viewer /islandora/object/uuid:513e4f13-1627-455a-89fa-0e53e0133892/datastream/OBJ/view