A Tailor-Made Deazaflavin-Mediated Recycling System for Artificial Nicotinamide Cofactor Biomimetics

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 F 420 :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 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 ( Ts OYE). 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 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.

Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform

“…4B ), a Total Turnover Frequency of ~117 turnovers per hour averaged over the 16 day reaction. This represents the highest TTN value reported to date for a noncanonical cofactor cycling system 16 , 46 – 49 , and it approaches the TTN values reported for the wild-type PTDH enzyme with NAD + (80,000) 50 . In addition to its robust temporal stability, the low K M of LY-13 with NMN + (Table 1 ) has been shown to be advantageous (Fig.…”

Ene‐Reductase: A Multifaceted Biocatalyst in Organic Synthesis

“…Over the years, several studies have been carried out in search of alternative hydride sources, [21,22] and likewise, several new approaches have been employed for the cofactor regeneration process. [7] Among them, nicotinamide biomimetics approaches, [23][24][25][26][27][28] protein engineering approaches, [29,30] and coenzyme-independent FMN reduction processes [31] are particularly very important.…”

Design of fusion enzymes for biocatalytic applications in aqueous and non-aqueous media

“…Although oxidoreductasesmediated selective oxidation is considered one of the most important transformations in organic chemistry, studies on oxidoreductases (EC 1) are still limited (Hollmann et al, 2011;Ringborg et al, 2017;Dong et al, 2018). Between 2000 and 2015, 68% of the patents covering biocatalytic applications were based on oxidoreductases, despite the fact that they account for "only" one-third approximately of all the known enzymes (Drenth et al, 2021). The limited use of oxidoreductase at technical scales in organic media is not only due to the instability of redox enzymes per se but also the fact that up to 50% approximately of known oxidoreductases are cofactor-dependent [NAD(P)H].…”