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Literature summary for 1.11.1.16 extracted from
- The making of versatile peroxidase by directed evolution (2018), Biocatal. Biotransform., 36, 1-11 .
No PubMed abstract available
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| all attempts to express a functional versatile peroxidase as soluble protein in Escherichia coli fail, recombinant enzyme expression and secretion from Saccharomyces cerevisiae, overexpression of enzyme mutant E37K/V160A/T184M/Q202L in Pichia pastoris | Pleurotus eryngii |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| D22N/T45A/E83G/I103V/G107S/P141A/F186L | site-directed mutagenesis | Pleurotus eryngii |
| E140G/P182S/Q229P | site-directed mutagenesis, the mutant BB-8 is active over an enhanced pH range compared to wild-type and displays strong hyperactivation after incubation at alkaline pH with a 3fold increase in activity, The active pH range for mutant BB-8 is expanded considerably for several substrates, including ABTS, sinapic acid and guaiacol. Consequently, BB-8 is active in the acid range (pH 3-4) and remarkably, in the pH interval from 5 to 9 in which the activity of the parental VP is negligible. The kinetic parameters measured for ABTS reveals enhanced catalytic efficiency at acid pH as result of increased affinity, which permits BB-8 to remain active at basic pHs. This effect is mostly attributed to the E140G mutation that enables the mutant to work with similar catalytic efficiency at pH 6 as the parental type at pH 3.5, due to the widening of the heme channel. Whilst the activity against Mn2+ is diminished due to the P182S mutation introduced close to this catalytic site, this mutation offers the first experimental insight into the role of the Mn2+ site for the direct (non-mediated) oxidation of ABTS at neutral/basic pH | Pleurotus eryngii |
| E37K/H39R/V160A/T184M/Q202L/D213A/G330R | site-directed mutagenesis of enzyme mutant E37K/V160A/T184M/Q202L introducing three additional stabilizing point mutations, the final mutant (2-1B) shows an overall enhancement of 8°C in kinetic thermostability compared to wild-type enzyme, the specific activity increases 2.5fold, and the expression rate is enhanced by 52 fold. The thermostability mutant 2-1B displays remarkable stability at alkaline pH (with a residual activity above 60% at pH 9 after 120 h of incubation), which is rather unusual in fungal peroxidases. Although 2-1B is stable at alkaline conditions, there is hardly any activity at its three catalytic sites at basic pH | Pleurotus eryngii |
| E37K/V160A/T184M/Q202L | site-directed mutagenesis, the secretion of the mutant enzyme from recombinant Saccharomyces cerevisiae improves 129fold compared to wild-type, yielding 22 mg/l of active, soluble and stable enzyme, overexpression in Pichia pastoris, the enzyme is secreted | Pleurotus eryngii |
| additional information | due to its broad substrate scope and minor requirements, versatile peroxidase is an extremely attractive blueprint to be designed by the directed evolution tool-box, directed evolution for functional expression in Saccharomyces cerevisiae, directed evolution for activity at alkaline pH, overview | Pleurotus eryngii |
| N11D/G35K/E40K/T45A/S86R/P141A/F186L/T323I | site-directed mutagenesis | Pleurotus eryngii |
| W164X | site-directed mutagenesis, no activity at the catalytic Trp164 at basic pH due to the fact that the reduction potential of the Trp164 radical decreases as the pH increases, hindering the oxidation of high-redox potential substrates at neutral/basic pH. The long-range electron transfer pathway from Trp164 to the heme is permanently cancelled out at pHs above pH 5.0, thereby diverting the oxidative route for the oxidation of low-redox potential substrates to the other two catalytic sites at the time that the oxidation of high-redox potential compounds is supressed | Pleurotus eryngii |
Localization
| Localization | Comment | Organism | GeneOntology No. | Textmining |
|---|---|---|---|---|
| extracellular | the enzyme is secreted | Pleurotus eryngii | - |
- |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Ca2+ | required, binding structure, three-dimensional modeling, overview | Pleurotus eryngii |  |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 2 Mn(II) + 2 H+ + H2O2 | Pleurotus eryngii | - |
2 Mn(III) + 2 H2O | - |
? | |
| Reactive Black 5 + 2 H+ + H2O2 | Pleurotus eryngii | - |
oxidized Reactive Black 5 + 2 H2O | - |
? | |
| veratryl alcohol + H2O2 | Pleurotus eryngii | - |
verytryl aldehyde + 2 H2O | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Pleurotus eryngii | O94753 | - |
- |
Reaction
| Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|
| 2 manganese(II) + 2 H+ + H2O2 = 2 manganese(III) + 2 H2O | at the ferric resting state, heme peroxidases are first activated by a single molecule of H2O2 to yield an oxidized catalytic intermediate called compound I (oxoferryl IV porphyrin p-cation radical), releasing one molecule of water as the only by-product. The enzyme then catalyzes two consecutive one-electron oxidations of two reducing substrates, regenerating the ground reduced state through a second catalytic intermediate called compound II (oxoferryl) and with the concomitant production of a second molecule of water. The main limiting step within this catalytic cycle is the low conversion rate from compound II to the ground state, which allows the former to accumulate and react with a new molecule of H2O2, either in the presence (with an excess of H2O2) or absence (at catalytic concentrations of H2O2) of the reducing substrate | Pleurotus eryngii |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 2 Mn(II) + 2 H+ + H2O2 | - |
Pleurotus eryngii | 2 Mn(III) + 2 H2O | - |
? | |
| 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) + 2 H+ + H2O2 | - |
Pleurotus eryngii | oxidized 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) + 2 H2O | - |
? | |
| guaiacol + H2O2 | - |
Pleurotus eryngii | oxidized guaiacol + 2 H2O | - |
? | |
| Reactive Black 5 + 2 H+ + H2O2 | - |
Pleurotus eryngii | oxidized Reactive Black 5 + 2 H2O | - |
? | |
| sinapic acid + H2O2 | - |
Pleurotus eryngii | ? + 2 H2O | - |
? | |
| veratryl alcohol + H2O2 | - |
Pleurotus eryngii | verytryl aldehyde + 2 H2O | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| Vpl2 | - |
Pleurotus eryngii |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| heme b | - |
Pleurotus eryngii | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| additional information | versatile peroxidase (VP) has an access channel that is open to the solvent and where low-redox potential substrates are oxidized. In addition, VP has a superficial catalytic tryptophan that, in its active state, oxidizes both low-redox and more significantly, high redox potential substrates through a long-range electron transfer pathway to the heme, like lignin peroxidase. In the sagittal plane of the protein structure there is a small heme access channel where Mn2+ is oxidized to Mn3+, the latter acting as a diffusible oxidizer as also occurs in MnP (EC 1.11.1.13) | Pleurotus eryngii |
| physiological function | versatile peroxidase (VP) secreted by white-rot fungi is involved in the degradation of lignin within land ecosystems, with a broad substrate scope and minor requirements | Pleurotus eryngii |
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