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Analysis of Agaricus meleagris pyranose dehydrogenase N-glycosylation sites and performance of partially non-glycosylated enzymes

Gonaus, Christoph; Maresch, Daniel; Schropp, Katharina; Ó Conghaile, Peter; Leech, Dónal; Gorton, Lo LU and Peterbauer, Clemens K (2017) In Enzyme and Microbial Technology 99. p.57-66
Abstract

Pyranose Dehydrogenase 1 from the basidiomycete Agaricus meleagris (AmPDH1) is an oxidoreductase capable of oxidizing a broad variety of sugars. Due to this and its ability of dioxidation of substrates and no side production of hydrogen peroxide, it is studied for use in enzymatic bio-fuel cells. In-vitro deglycosylated AmPDH1 as well as knock-out mutants of the N-glycosylation sites N75 and N175, near the active site entrance, were previously shown to improve achievable current densities of graphite electrodes modified with AmPDH1 and an osmium redox polymer acting as a redox mediator, up to 10-fold. For a better understanding of the role of N-glycosylation of AmPDH1, a systematic set of N-glycosylation site... (More)

Pyranose Dehydrogenase 1 from the basidiomycete Agaricus meleagris (AmPDH1) is an oxidoreductase capable of oxidizing a broad variety of sugars. Due to this and its ability of dioxidation of substrates and no side production of hydrogen peroxide, it is studied for use in enzymatic bio-fuel cells. In-vitro deglycosylated AmPDH1 as well as knock-out mutants of the N-glycosylation sites N75 and N175, near the active site entrance, were previously shown to improve achievable current densities of graphite electrodes modified with AmPDH1 and an osmium redox polymer acting as a redox mediator, up to 10-fold. For a better understanding of the role of N-glycosylation of AmPDH1, a systematic set of N-glycosylation site mutants was investigated in this work, regarding expression efficiency, enzyme activity and stability. Furthermore, the site specific extend of N-glycosylation was compared between native and recombinant wild type AmPDH1. Knocking out the site N252 prevented the attachment of significantly extended N-glycan structures as detected on polyacrylamide gel electrophoresis, but did not significantly alter enzyme performance on modified electrodes. This suggests that not the molecule size but other factors like accessibility of the active site improved performance of deglycosylated AmPDH1/osmium redox polymer modified electrodes. A fourth N-glycosylation site of AmPDH1 could be confirmed by mass spectrometry at N319, which appeared to be conserved in related fungal pyranose dehydrogenases but not in other members of the glucose-methanol-choline oxidoreductase structural family. This site was shown to be the only one that is essential for functional recombinant expression of the enzyme.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Agaricus meleagris, Enzymatic biofuel cell, N-glycosylation, Pyranose dehydrogenase
in
Enzyme and Microbial Technology
volume
99
pages
10 pages
publisher
Elsevier
external identifiers
  • scopus:85010904396
  • wos:000395218500008
ISSN
0141-0229
DOI
10.1016/j.enzmictec.2017.01.008
language
English
LU publication?
yes
id
43cc9e8c-492d-4c5e-a696-0cc2f77fc1bf
date added to LUP
2017-02-14 08:44:53
date last changed
2018-01-07 11:49:34
@article{43cc9e8c-492d-4c5e-a696-0cc2f77fc1bf,
  abstract     = {<p>Pyranose Dehydrogenase 1 from the basidiomycete Agaricus meleagris (AmPDH1) is an oxidoreductase capable of oxidizing a broad variety of sugars. Due to this and its ability of dioxidation of substrates and no side production of hydrogen peroxide, it is studied for use in enzymatic bio-fuel cells. In-vitro deglycosylated AmPDH1 as well as knock-out mutants of the N-glycosylation sites N<sup>75</sup> and N<sup>175</sup>, near the active site entrance, were previously shown to improve achievable current densities of graphite electrodes modified with AmPDH1 and an osmium redox polymer acting as a redox mediator, up to 10-fold. For a better understanding of the role of N-glycosylation of AmPDH1, a systematic set of N-glycosylation site mutants was investigated in this work, regarding expression efficiency, enzyme activity and stability. Furthermore, the site specific extend of N-glycosylation was compared between native and recombinant wild type AmPDH1. Knocking out the site N<sup>252</sup> prevented the attachment of significantly extended N-glycan structures as detected on polyacrylamide gel electrophoresis, but did not significantly alter enzyme performance on modified electrodes. This suggests that not the molecule size but other factors like accessibility of the active site improved performance of deglycosylated AmPDH1/osmium redox polymer modified electrodes. A fourth N-glycosylation site of AmPDH1 could be confirmed by mass spectrometry at N<sup>319</sup>, which appeared to be conserved in related fungal pyranose dehydrogenases but not in other members of the glucose-methanol-choline oxidoreductase structural family. This site was shown to be the only one that is essential for functional recombinant expression of the enzyme.</p>},
  author       = {Gonaus, Christoph and Maresch, Daniel and Schropp, Katharina and Ó Conghaile, Peter and Leech, Dónal and Gorton, Lo and Peterbauer, Clemens K},
  issn         = {0141-0229},
  keyword      = {Agaricus meleagris,Enzymatic biofuel cell,N-glycosylation,Pyranose dehydrogenase},
  language     = {eng},
  month        = {04},
  pages        = {57--66},
  publisher    = {Elsevier},
  series       = {Enzyme and Microbial Technology},
  title        = {Analysis of Agaricus meleagris pyranose dehydrogenase N-glycosylation sites and performance of partially non-glycosylated enzymes},
  url          = {http://dx.doi.org/10.1016/j.enzmictec.2017.01.008},
  volume       = {99},
  year         = {2017},
}