An integrated view of redox and catalytic properties of B-type PpDyP from Pseudomonas putida MET94 and its distal variants

Mendes, Sónia; Brissos, Vânia; Gabriel, Antonieta; Catarino, Teresa; Turner, David L.; Todorović, Smilja; Martins, Lı́gia O.

doi:10.1016/j.abb.2015.03.009

Cited by 42 publications

(72 citation statements)

References 41 publications

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“…Mn 2+ ). 23 The pH opt of PpDyP is 5 (for ABTS as a substrate). 20 RR spectra of resting state PpDyP measured at pH 7.6, show broad and asymmetric n 3 band indicative of multiple species, Fig.…”

Section: B-type Dypsmentioning

confidence: 99%

“…16,21 The roles of conserved distal residues, Asp132, Asn136 and Arg214, in the catalytic mechanism of PpDyP were addressed by generation of site-directed variants, and the mutation-induced alterations of the active site were evaluated by RR. 23 It was demonstrated that the distal Arg in particular plays an essential role in PpDyP, as in the case of DyPB from Rhodococcus jostii RHA1 but not in DyPs from the A and D subfamilies. The D132N and N136L mutations have a substantial effect on the geometry of the distal side of the active site.…”

Section: B-type Dypsmentioning

confidence: 99%

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Resonance Raman view of the active site architecture in bacterial DyP-type peroxidases

et al. 2020

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Section: B-type Dypsmentioning

confidence: 99%

Section: B-type Dypsmentioning

confidence: 99%

Resonance Raman view of the active site architecture in bacterial DyP-type peroxidases

et al. 2020

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“…For Thermomonospora curvata DyP second-order rate constants of k = 5.9 x 10 6 M -1 s -1 (pH 7.8) and 4.0 x 10 M -1 s -1 (pH 3.0) have been determined 12 and from Thermobifida fusca k = 5.5 x 10 4 M -1 s -1 (pH 5.5) 22 . Elsewhere, only two B-type DyPs have had their compound I kinetics determined with k = 1.8 x10 5 M -1 s -1 (pH 7.5) for the DyP from Rhodococcus jostii 9 and k = 1.4 x 10 6 M -1 s -1 (pH 7.0) for the DyP from Pseudomonas putida 23 . Thus, a significant variation in compound I formation within the DyP family and within sub-families is apparent, with the absence of data on types C and D notable exceptions, highlighting the need for further kinetic studies to build a complete picture of the catalytic properties of this class of haem peroxidase.…”

Section: Spectral Characterisation and Kinetics Of Dtpa Compound I Fomentioning

confidence: 99%

Kinetic characterisation of a dye decolourising peroxidase from Streptomyces lividans: new insight into the mechanism of anthraquinone dye decolourisation

2017

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Dye decolourising peroxidases are the most recent family of haem peroxidases to be discovered. The oxidising potential of these enzymes is driven by the formation of ferryl intermediates that enables them to oxidise synthetic dye molecules that are widely used in the textile industry. We have investigated the catalytic cycle of a dye decolourising peroxidase (DtpA) from a biotechnologically important bacterium Streptomyces lividans. Using a combination of steady-state and stopped-flow kinetic investigations, we have determined the rate constants for all steps in the catalytic cycle with a range of substrate molecules. For most substrates, the value of k/K measured by steady-state kinetics is equal to the slowest step in catalysis measured by stopped-flow spectroscopy, namely the decay of the ferryl Fe[double bond, length as m-dash]O species (compound II) to form the ferric species. With the anthraquinone-based dye, reactive blue 19 (RB19) unusual steady-state kinetic behaviour is observed, which we propose through kinetic modelling of the catalytic cycle is due to a disproportionation mechanism of the dye. At low RB19 concentrations, the rate of disproportionation is slower than that of the rate determining step in DtpA, whereas at higher concentrations of RB19 the rate of disproportionation is faster. This mechanism obviates the need to postulate secondary sites for substrate binding on the enzyme which has been previously proposed for other dye decolourising haem peroxidases.

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“…Raman spectroscopy is also an efficient technique that is capable of investigating protein molecular structures, good for solid samples as well as aqueous solutions [24]. Recently, Raman spectra have been used for the analysis of different structures of various proteins [24, 52, 53]. Therefore, in the present study, comparisons of major Raman scattering bands were made between soluble and refolded MnP.…”

Section: Discussionmentioning

confidence: 99%

Expression of a fungal manganese peroxidase in Escherichia coli: a comparison between the soluble and refolded enzymes

et al. 2016

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BackgroundManganese peroxidase (MnP) from Irpex lacteus F17 has been shown to have a strong ability to degrade recalcitrant aromatic pollutants. In this study, a recombinant MnP from I. lacteus F17 was expressed in Escherichia coli Rosetta (DE3) in the form of inclusion bodies, which were refolded to achieve an active enzyme. Further, we optimized the in vitro refolding conditions to increase the recovery yield of the recombinant protein production. Additionally, we attempted to express recombinant MnP in soluble form in E. coli, and compared its activity with that of refolded MnP.ResultsRefolded MnP was obtained by optimizing the in vitro refolding conditions, and soluble MnP was produced in the presence of four additives, TritonX-100, Tween-80, ethanol, and glycerol, through incubation at 16 °C. Hemin and Ca2+ supplementation was crucial for the activity of the recombinant protein. Compared with refolded MnP, soluble MnP showed low catalytic efficiencies for Mn2+ and H2O2 substrates, but the two enzymes had an identical, broad range substrate specificity, and the ability to decolorize azo dyes. Furthermore, their enzymatic spectral characteristics were analysed by circular dichroism (CD), electronic absorption spectrum (UV-VIS), fluorescence and Raman spectra, indicating the differences in protein conformation between soluble and refolded MnP. Subsequently, size exclusion chromatography (SEC) and dynamic light scattering (DLS) analyses demonstrated that refolded MnP was a good monomer in solution, while soluble MnP predominantly existed in the oligomeric status.ConclusionsOur results showed that two forms of recombinant MnP could be expressed in E. coli by varying the culture conditions during protein expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0317-2) contains supplementary material, which is available to authorized users.

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An integrated view of redox and catalytic properties of B-type PpDyP from Pseudomonas putida MET94 and its distal variants

Cited by 42 publications

References 41 publications

Resonance Raman view of the active site architecture in bacterial DyP-type peroxidases

Resonance Raman view of the active site architecture in bacterial DyP-type peroxidases

Kinetic characterisation of a dye decolourising peroxidase from Streptomyces lividans: new insight into the mechanism of anthraquinone dye decolourisation

Expression of a fungal manganese peroxidase in Escherichia coli: a comparison between the soluble and refolded enzymes

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