New function of aldoxime dehydratase: Redox catalysis and the formation of an expected product

Abstract

In general, hemoproteins are capable of catalyzing redox reactions. Aldoxime dehydratase (OxdA), which is a unique heme-containing enzyme, catalyzes the dehydration of aldoximes to the corresponding nitriles. Its reaction is a rare example of heme directly activating an organic substrate, unlike the utilization of H2O2 or O2 as a mediator of catalysis by other heme-containing enzymes. While it is unknown whether OxdA catalyzes redox reactions or not, we here for the first time detected catalase activity (which is one of the redox activities) of wild-type OxdA, OxdA(WT). Furthermore, we constructed a His320 → Asp mutant of OxdA [OxdA(H320D)], and found it exhibits catalase activity. Determination of the kinetic parameters of OxdA(WT) and OxdA(H320D) revealed that their Km values for H2O2 were similar to each other, but the kcat value of OxdA(H320D) was 30 times higher than that of OxdA(WT). Next, we examined another redox activity and found it was the peroxidase activity of OxdAs. While both OxdA(WT) and OxdA(H320D) showed the activity, the activity of OxdA(H320D) was dozens of times higher than that of OxdA(WT). These findings demonstrated that the H320D mutation enhances the peroxidase activity of OxdA. OxdAs (WT and H320D) were found to catalyze another redox reaction, a peroxygenase reaction. During this reaction of OxdA(H320D) with 1-methoxynaphthalene as a substrate, surprisingly, the reaction mixture changed to a color different from that with OxdA(WT), which was due to the known product, Russig’s blue. We purified and identified the new product as 1-methoxy-2-naphthalenol, which has never been reported as a product of the peroxygenase reaction, to the best of our knowledge. These findings indicated that the H320D mutation not only enhanced redox activities, but also significantly altered the hydroxylation site of the substrate.

Nitrile biosynthesis in nature: how and why?

“…158 Furthermore, the versatile OxdA also functions as catalase, peroxidase, and peroxygenase, revealing its catalytic promiscuity. 169…”

“…158 Furthermore, the versatile OxdA also functions as catalase, peroxidase, and peroxygenase, revealing its catalytic promiscuity. 169 Six new bacterial aldoxime dehydratases were discovered recently by using 3DM system (a soware suite for protein superfamily analysis) based on the well-studied OxdB, namely OxdVP from Variovorax paradoxus, OxdHsp from Hydrogenophaga sp. RAC07, OxdPsp from Pseudomonas sp.…”

A Unique P450 Peroxygenase System Facilitated by a Dual-Functional Small Molecule: Concept, Application, and Perspective

“…The non-natural DFSM-facilitated P450-H 2 O 2 system described above mainly catalyzes various per-oxygenation reactions, including epoxidation, hydroxylation, and sulfoxidation [66,[92][93][94][95]. Interestingly, the oxidation of guaiacol, a classical substrate of peroxidases [141][142][143][144], catalyzed by the DFSM-facilitated P450BM3-H 2 O 2 system yielded demethylated catechol as a major product, suggesting it mainly functioned as a peroxygenase but not as a peroxidase [94]. After carefully analyzing the catalytic mechanism of the potential competitive oxidation pathways in the DFSM-facilitated P450BM3-H 2 O 2 system, Ma et al hypothesized that mutation of redox-sensitive residues may enable switching of peroxygenase activity to peroxidase activity [145].…”

Switching an Artificial P450 Peroxygenase into Peroxidase via Mechanism-Guided Protein Engineering

“…On the basis of the established mechanistic understanding of catalytic H 2 O 2 activation in natural peroxidases and peroxygenases, our group has recently used a modified fatty acid hydroxylase protein from Bacillus megaterium (P450BM3) to construct an artificial P450-H 2 O 2 system by installing a rationally designed dual-functional small molecule (DFSM) as a built-in base to assist with H 2 O 2 activation (Figure A). − The system efficiently catalyzed various peroxygenation reactions, including epoxidations, sulfoxidations, and hydroxylations (Figure B). , However, catalytic guaiacol oxidation with this system generated demethylated catechol as the major product . Guaiacol is a classical substrate of peroxidases, − so this unusual result suggested that the DFSM-facilitated P450BM3-H 2 O 2 system mainly functioned as a peroxygenase but not as a peroxidase (Figure C).…”