Two Oxidation Sites for Low Redox Potential Substrates

Morales, M.P.; Mate, M.J.; Romero, Antonio; Martı́nez, Marı́a Jesús; Martı́nez, Ángel T.; Ruíz-Dueñas, Francisco J.

doi:10.1074/jbc.m112.405548

Cited by 69 publications

(44 citation statements)

References 39 publications

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“…Here, we purified the native enzyme and characterized the reaction mechanisms from GP medium. Three substrates, Mn 2ϩ , RB5, and OII, were studied, representing oxidation by the three sites of the enzyme: (i) the well-known Mn 2ϩ binding and active site (13), (ii) the tryptophanyl radical (Trp164) active site (18), and (iii) the heme edge active site (14), respectively. We studied the interactions among these sites and determined that Mn 2ϩ inhibited the direct oxidation of both RB5 and OII, even though this Table 4).…”

Section: Discussionmentioning

confidence: 99%

“…We selected three substrates to further characterize purified VP1, namely, Mn 2ϩ , RB5, and OII, each of which is known to be oxidized at a different site: the well-known Mn 2ϩ binding and active site (13), the tryptophanyl radical (Trp164) active site (18), and the heme edge active site (14,15), respectively.…”

Section: Figmentioning

confidence: 99%

“…The first is the exposed heme edge that provides VPs the capability to directly oxidize phenols, amines, and small dye compounds in the absence of Mn 2ϩ (14). This site is typical of some other peroxidases, such as LiP, Coprinus cinereus peroxidase (CiP), and horseradish peroxidase (HRP) (EC 1.11.1.7) (14)(15)(16)(17).…”

mentioning

confidence: 99%

“…This site is responsible for the direct oxidation of low-and high-redox-potential compounds, including those that LiP can oxidize only in the presence of redox mediators (14,18,19). It was suggested that this site acts as a substrate intermediate protein radical center and initiates a long-range electron transfer (LRET) pathway leading to the heme (15,20,21).…”

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confidence: 99%

“…P. ostreatus VP1 and P. eryngii VPL2, the most intensively studied ligninolytic enzyme, share 96% identity in their sequences. The crystal structures of these enzymes have been solved (2,14,24). The current study focuses on the Mn 2ϩ -independent reactions with regard to the complex versatile nature of P. ostreatus VP1.…”

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confidence: 99%

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Limits of Versatility of Versatile Peroxidase

Knop

Levinson

Makovitzki

et al. 2016

Appl Environ Microbiol

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Although Mn2؉ is the most abundant substrate of versatile peroxidases (VPs), repression of Pleurotus ostreatus vp1 expression occurred in Mn 2؉ -sufficient medium. This seems to be a biological contradiction. The aim of this study was to explore the mechanism of direct oxidation by VP1 under Mn 2؉ -deficient conditions, as it was found to be the predominant enzyme during fungal growth in the presence of synthetic and natural substrates. The native VP1 was purified and characterized using three substrates, Mn 2؉ , Orange II (OII), and Reactive Black 5 (RB5), each oxidized by a different active site in the enzyme. While the pH optimum for Mn 2؉ oxidation is 5, the optimum pH for direct oxidation of both dyes was found to be 3. Indeed, effective in vivo decolorization occurred in media without addition of Mn 2؉ only under acidic conditions. We have determined that Mn 2؉ inhibits in vitro the direct oxidation of both OII and RB5 while RB5 stabilizes both Mn 2؉ and OII oxidation. Furthermore, OII was found to inhibit the oxidation of both Mn 2؉ and RB5. In addition, we could demonstrate that VP1 can cleave OII in two different modes. Under Mn 2؉ -mediated oxidation conditions, VP1 was able to cleave the azo bond only in asymmetric mode, while under the optimum conditions for direct oxidation (absence of Mn 2؉ at pH 3) both symmetric and asymmetric cleavages occurred. We concluded that the oxidation mechanism of aromatic compounds by VP1 is controlled by Mn 2؉ and pH levels both in the growth medium and in the reaction mixture. IMPORTANCEVP1 is a member of the ligninolytic heme peroxidase gene family of the white rot fungus Pleurotus ostreatus and plays a fundamental role in biodegradation. This enzyme exhibits a versatile nature, as it can oxidize different substrates under altered environmental conditions. VPs are highly interesting enzymes due to the fact that they contain unique active sites that are responsible for direct oxidation of various aromatic compounds, including lignin, in addition to the well-known Mn 2؉ binding active site. This study demonstrates the limits of versatility of P. ostreatus VP1, which harbors multiple active sites, exhibiting a broad range of enzymatic activities, but they perform differently under distinct conditions. The versatility of P. ostreatus and its enzymes is an advantageous factor in the fungal ability to adapt to changing environments. This trait expands the possibilities for the potential utilization of P. ostreatus and other white rot fungi.

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Section: Discussionmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Limits of Versatility of Versatile Peroxidase

Knop

Levinson

Makovitzki

et al. 2016

Appl Environ Microbiol

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Engineering Cytochrome P450BM3 Enzymes for Direct Nitration of Unsaturated Hydrocarbons

et al. 2023

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Applications of the peroxidase activity of cytochrome P450 enzymes in synthetic chemistry remain largely unexplored. We present herein a protein engineering strategy to increase cytochrome P450BM3 peroxidase activity for the direct nitration of aromatic compounds and terminal aryl‐substituted olefins in the presence of a dual‐functional small molecule (DFSM). Site‐directed mutations of key active‐site residues allowed the efficient regulation of steric effects to limit substrate access and, thus, a significant decrease in monooxygenation activity and increase in peroxidase activity. Nitration of several phenol and aniline compounds also yielded ortho‐ and para‐nitration products with moderate‐to‐high total turnover numbers. Besides direct aromatic nitration by P450 variants using nitrite as a nitrating agent, we also demonstrated the use of the DFSM‐facilitated P450 peroxidase system for the nitration of the vinyl group of styrene and its derivatives.

show abstract

Engineering Cytochrome P450BM3 Enzymes for Direct Nitration of Unsaturated Hydrocarbons

et al. 2023

View full text Add to dashboard Cite

Applications of the peroxidase activity of cytochrome P450 enzymes in synthetic chemistry remain largely unexplored. We present herein a protein engineering strategy to increase cytochrome P450BM3 peroxidase activity for the direct nitration of aromatic compounds and terminal aryl-substituted olefins in the presence of a dual-functional small molecule (DFSM). Site-directed mutations of key active-site residues allowed the efficient regulation of steric effects to limit substrate access and, thus, a significant decrease in monooxygenation activity and increase in peroxidase activity. Nitration of several phenol and aniline compounds also yielded ortho-and para-nitration products with moderate-to-high total turnover numbers. Besides direct aromatic nitration by P450 variants using nitrite as a nitrating agent, we also demonstrated the use of the DFSM-facilitated P450 peroxidase system for the nitration of the vinyl group of styrene and its derivatives.

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Two Oxidation Sites for Low Redox Potential Substrates

Cited by 69 publications

References 39 publications

Limits of Versatility of Versatile Peroxidase

Limits of Versatility of Versatile Peroxidase

Engineering Cytochrome P450BM3 Enzymes for Direct Nitration of Unsaturated Hydrocarbons

Engineering Cytochrome P450BM3 Enzymes for Direct Nitration of Unsaturated Hydrocarbons

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