A Tailor-Made Deazaflavin-Mediated Recycling System for Artificial Nicotinamide Cofactor Biomimetics
Jeroen Drenth, Guang Yang, Caroline E. Paul, Marco W. Fraaije
Abstract
Nicotinamide adenine dinucleotide (NAD) and its 2phosphorylated 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 F 420 :NADPH oxidoreductase from Thermobifida fusca (Tf u-FNO), by structureinspired site-directed mutagenesis, to accommodate the unnatural N1 substituents of eight NCBs. The extraordinarily low redox potential of the natural cofactor F 420 H 2 was then exploited to reduce these NCBs. Wild-type enzyme had detectable activity toward all selected NCBs, with K m values in the millimolar range and k cat 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 k cat value of 4.2 s -1 toward 1-benzyl-3-acetylpyridine (BAP + ), which is similar to the k cat 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 F 420 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 Tf u-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.