Catalytic Promiscuity of Horseradish Peroxidase: Aerobic Oxidative Regeneration of Oxidized Nicotinamide Cofactors

Abstract

NAD(P)+ regeneration is of great importance for dehydrogenase (DH)‐catalyzed oxidations. In this work, we report horseradish peroxidase (HRP)‐catalyzed aerobic oxidation of the reduced nicotinamide cofactors, including natural and non‐natural ones, into the oxidized form at neutral and alkaline pH enabled by external amino acids (AAs). HRP showed promiscuous yet extremely low oxidase activity toward NADH at pH 7 in the absence of both AAs and mediators, as evidenced by the observation of compound III. The mechanistic study revealed that AAs as decoy molecules (likely act as acid‐base catalysts) and mediators significantly promoted the formation of compound III as well as its decay back to the ground state. The applicability of the aerobic cofactor regeneration method was demonstrated by the HRP total turnover number up to 88 000 as well as high yields (91–99 %) in bi‐enzymatic synthesis of carboxylic acids. The present work offers an alternative method to recycle NAD(P)+. Also, it may open up possibilities for HRP‐catalyzed aerobic oxidations.

Revisiting Horseradish Peroxidase-Catalyzed Aerobic Oxidation of NAD(P)H in the Presence of Mn(II) and Mediators: Application in Catalytic Oxidation

“…46 Intermediate compound I was not observed in this work, likely due to its labile nature. In terms of the mechanism, therefore, aerobic NADH oxidation by the HRP/ Mn 2+ /mediator system is distinct from that by the HRP/AA/ mediator system reported recently by us, 31 as oxidase intermediate compound III instead of compound I/II is involved in the latter. The distinctions between the two systems may be partially rationalized by their catalytic mechanisms.…”

“…More than 60 years ago, several groups reported that NAD­(P)H was aerobically oxidized by HRP in the presence of Mn 2+ and mediators. − In this work, the “old” chemobiocatalytic NAD­(P) + oxidation reaction was revisited along with the expansion of its substrate scope; a putative mechanism was proposed on the basis of the results obtained herein and previous literature. Notably, the efficiency of the present system involving Mn 2+ is much higher than that of a recent system involving AAs, evidenced by the reaction periods required for almost complete substrate conversion at the same substrate concentration (approximately 1 vs 25 min). More importantly, the application potential of this system was demonstrated in DH-catalyzed (cascade) oxidation and photobiocatalysis.…”

“… − Unfortunately, the suicide inactivation of the enzyme in the presence of excess H 2 O 2 greatly impedes its wide application in synthetic chemistry. , It is envisaged that HRP-catalyzed aerobic oxidation may create new possibilities. As a matter of fact, the fundamental biochemistry and biophysics studies on the peroxidase–oxidase oscillatory oxidation of NAD­(P)­H, indoleacetic acid, and dihydroxyfumaric acid with O 2 by HRP may date back to the early 1960s. − To our knowledge, its synthetic application in aerobic oxidation has not been demonstrated until very recently by us (Scheme ), where HRP enabled the aerobic oxidation of NAD­(P)H and synthetic mimics in the presence of external amino acids (AAs) and mediators. HRP was activated into a reactive intermediate compound III (evidenced by the appearance of two characteristic peaks at 544 and 579 nm at the Q-band) by O 2 in the presence of two external additives, which oxidized the mediator phenol into a phenoxy radical, together with the release of the native enzyme.…”

“…28−30 To our knowledge, its synthetic application in aerobic oxidation has not been demonstrated until very recently by us (Scheme 1), 31 where HRP enabled the aerobic oxidation of NAD(P)H and synthetic mimics in the presence of external amino acids (AAs) and mediators. HRP was activated into a reactive intermediate compound III (evidenced by the appearance of two characteristic peaks at 544 and 579 nm at the Q-band) by O 2 in the presence of two external additives, 31 which oxidized the mediator phenol into a phenoxy radical, together with the release of the native enzyme. Although a proof of concept was demonstrated, the efficiency of chemoenzymatic NAD(P)H oxidation remained very low.…”

One-pot photoenzymatic synthesis of maleic acid and its derivatives from bio-based furfural via catalytic cascades

“…42 Recently, an in vitro stable ALDH from Pseudomonas aeruginosa ( Pa ALDH) was identified by our group, and it showed good activities toward a variety of aromatic aldehydes. 58 To test whether the enzyme is tolerant toward photocatalysis inactivation, Pa ALDH was merged with EY@NKA for photoenzymatic oxidation of furfural (the first step in Scheme 2), 59 in which EY@NKA served as a catalyst for both photocatalytic NADP + recycling and FCA photooxygenation. Fig.…”