Activity assays of methanol dehydrogenases

Gutenthaler, Sophie M.; Phi, Manh Tri; Singer, Helena; Daumann, Lena J.

doi:10.1016/bs.mie.2021.01.045

Cited by 3 publications

(9 citation statements)

References 55 publications

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“…The activity assay with cyt c GJ and artificial electron acceptors are based on the protocols reported by Gutenthaler et al [8] The enzymatic activity of MDH was assessed with native cyt c GJ through the reduction of equine heart cyt c (Sigma, CAS 9007-43-6). The reaction was monitored with an Epoch2 plate reader (formerly BioTek, now Agilent) at 45 °C through the increase of A 550 .…”

Section: Activity Assaysmentioning

confidence: 99%

“…Usually a pH of 9, additives and activators like ammonia, glycine ethyl ester, potassium cyanide and an excess of Ln are included in the assay mixture to optimize the conditions for the assays (these additives are not necessary for MDH from M. fumariolicum SolV). [8] These experimental conditions rarely reflect the physiological conditions inside the cell and are truly artificial. Furthermore, the source of the endogenous substrate that causes background reaction is still unknown.…”

Section: Introductionmentioning

confidence: 99%

“…The reduction of DCPIP results in its discoloration which is measured at 600 nm by UV/Vis. Based on previous work by Anthony and co‐workers, protein‐coupled activity assays for MDH from SolV using its physiological cyt c GJ partner were developed [8,15,19] . Commercially available cyt c from equine or bovine heart is used as secondary electron acceptor [15] .…”

Section: Introductionmentioning

confidence: 99%

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Assessing Lanthanide‐Dependent Methanol Dehydrogenase Activity: The Assay Matters

Phi,

Singer,

Zäh

et al. 2024

ChemBioChem

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Artificial dye‐coupled assays have been widely adopted as a rapid and convenient method to assess the activity of methanol dehydrogenases (MDH). Lanthanide(Ln)‐dependent XoxF‐MDHs are able to incorporate different lanthanides (Lns) in their active site. Dye‐coupled assays showed that the earlier Lns exhibit a higher enzyme activity than the late Lns. Despite widespread use, there are limitations: oftentimes a pH of 9 and activators are required for the assay. Moreover, Ln‐MDH variants are not obtained by isolation from the cells grown with the respective Ln, but by incubation of an apo‐MDH with the Ln. Herein, we report the cultivation of Ln‐dependent methanotroph Methylacidiphilum fumariolicum SolV with nine different Lns, the isolation of the respective MDHs and the assessment of the enzyme activity using the dye‐coupled assay. We compare these results with a protein‐coupled assay using its physiological electron acceptor cytochrome cGJ (cyt cGJ). Depending on the assay, two distinct trends are observed among the Ln series. The specific enzyme activity of La‐, Ce‐ and Pr‐MDH, as measured by the protein‐coupled assay, exceeds that measured by the dye‐coupled assay. This suggests that early Lns also have a positive effect on the interaction between XoxF‐MDH and its cyt cGJ thereby increasing functional efficiency.

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Section: Activity Assaysmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Assessing Lanthanide‐Dependent Methanol Dehydrogenase Activity: The Assay Matters

Phi,

Singer,

Zäh

et al. 2024

ChemBioChem

View full text Add to dashboard Cite

show abstract

“…Based on previous work by Anthony and co-workers, protein-coupled activity assays for MDH from strain SolV using its physiological cyt c GJ partner were developed. 30, 35, 40 Commercially available cyt c from equine or bovine heart is used as secondary electron acceptor. 35 Again, the reduction of the secondary electron acceptor can be monitored to determine the enzyme activity (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…Usually a pH of 9, additives and activators like ammonia, glycine ethyl ester, potassium cyanide and an excess of Lns are included in the assay mixture to optimize the conditions for the assays (these additives are not necessary for MDH from Methylacidiphilum fumariolicum SolV). 30 These experimental conditions rarely reflect the physiological conditions inside the cell and are truly artificial. Furthermore, the source of the endogenous substrate that causes background reaction is still unknown.…”

Section: Introductionmentioning

confidence: 99%

Assessing Lanthanide-Dependent Methanol Dehydrogenase Activity: The Assay Matters

Phi,

Singer,

Zäh

et al. 2023

Preprint

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Artificial dye-coupled assays have been widely adopted as a rapid and convenient method to assess the activity of methanol dehydrogenases (MDH). Lanthanide(Ln)-dependent XoxF-MDHs from methanotrophs and methylotrophs are able to incorporate different lanthanides (Lns) in their active site. The artificial dye-coupled assay showed that the earlier Lns exhibit a higher enzyme activity than the late Lns. Although this assay is widely used, there are limitations. It is not unusual that a pH of 9 is required and that activators like ammonium have to be added to the assay mixture which do not reflect the conditions inside the cell. Moreover, different Ln-MDH variants are not obtained by the direct isolation from the cells grown with the respective Ln, but by metal titration of the Ln in a partial-apo-MDH or by incubation of an apo-MDH with the Ln. Herein, we report the cultivation of Ln-dependent methanotroph Methylacidiphilum fumariolicum SolV with nine different Lns, the isolation of the respective MDH and the assessment of the enzyme activity using the artificial dye-coupled assay. We compare these results with an adapted protein-coupled assay using the physiological partner and electron acceptor cytochrome GJ (cyt cGJ) instead of artificial dyes. We demonstrate that, depending on the assay, two distinct trends are observed among the Ln series. The specific activity of La-, Ce- and Pr-MDH, as measured by the protein-coupled assay, exceeds the specific enzyme activity measured by the dye coupled assay. This suggests that the early Lns also have a positive effect on the interaction between XoxF-MDH and its cyt cGJ thereby increasing functional efficiency.

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Artificial multi-enzyme cascades and whole-cell transformation for bioconversion of C1 compounds: Advances, challenge and perspectives

Qiao,

Ma,

Zhang

et al. 2023

Synthetic and Systems Biotechnology

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Activity assays of methanol dehydrogenases

Cited by 3 publications

References 55 publications

Assessing Lanthanide‐Dependent Methanol Dehydrogenase Activity: The Assay Matters

Assessing Lanthanide‐Dependent Methanol Dehydrogenase Activity: The Assay Matters

Assessing Lanthanide-Dependent Methanol Dehydrogenase Activity: The Assay Matters

Artificial multi-enzyme cascades and whole-cell transformation for bioconversion of C1 compounds: Advances, challenge and perspectives

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