Analysis of N-glycosylation in fungal l-amino acid oxidases expressed in the methylotrophic yeast Pichia pastoris

Heß M, Grollius M, Duhay V, Koopmeiners S, Bloess S, Fischer von Mollard G (2021)
MicrobiologyOpen 10(4): e1224.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
 
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Abstract / Bemerkung
l-amino acid oxidases (LAAOs) catalyze the oxidative deamination of l-amino acids to corresponding alpha-keto acids. Here, we describe the heterologous expression of four fungal LAAOs in Pichia pastoris. cgLAAO1 from Colletotrichum gloeosporioides and ncLAAO1 from Neurospora crassa were able to convert substrates not recognized by recombinant 9His-hcLAAO4 from the fungus Hebeloma cylindrosporum described earlier thereby broadening the substrate spectrum for potential applications. 9His-frLAAO1 from Fibroporia radiculosa and 9His-laLAAO2 from Laccaria amethystine were obtained only in low amounts. All four enzymes were N-glycosylated. We generated mutants of 9His-hcLAAO4 lacking N-glycosylation sites to further understand the effects of N-glycosylation. All four predicted N-glycosylation sites were glycosylated in 9His-hcLAAO4 expressed in P.pastoris. Enzymatic activity was similar for fully glycosylated 9His-hcLAAO4 and variants without one or all N-glycosylation sites after acid activation of all samples. However, activity without acid treatment was low in a variant without N-glycans. This was caused by the absence of a hypermannosylated N-glycan on asparagine residue N54. The lack of one or all of the other N-glycans was without effect. Our results demonstrate that adoption of a more active conformation requires a specific N-glycosylation during biosynthesis. © 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
Erscheinungsjahr
2021
Zeitschriftentitel
MicrobiologyOpen
Band
10
Ausgabe
4
Art.-Nr.
e1224
eISSN
2045-8827
Page URI
https://pub.uni-bielefeld.de/record/2957226

Zitieren

Heß M, Grollius M, Duhay V, Koopmeiners S, Bloess S, Fischer von Mollard G. Analysis of N-glycosylation in fungal l-amino acid oxidases expressed in the methylotrophic yeast Pichia pastoris. MicrobiologyOpen. 2021;10(4): e1224.
Heß, M., Grollius, M., Duhay, V., Koopmeiners, S., Bloess, S., & Fischer von Mollard, G. (2021). Analysis of N-glycosylation in fungal l-amino acid oxidases expressed in the methylotrophic yeast Pichia pastoris. MicrobiologyOpen, 10(4), e1224. https://doi.org/10.1002/mbo3.1224
Heß, M., Grollius, M., Duhay, V., Koopmeiners, S., Bloess, S., and Fischer von Mollard, G. (2021). Analysis of N-glycosylation in fungal l-amino acid oxidases expressed in the methylotrophic yeast Pichia pastoris. MicrobiologyOpen 10:e1224.
Heß, M., et al., 2021. Analysis of N-glycosylation in fungal l-amino acid oxidases expressed in the methylotrophic yeast Pichia pastoris. MicrobiologyOpen, 10(4): e1224.
M. Heß, et al., “Analysis of N-glycosylation in fungal l-amino acid oxidases expressed in the methylotrophic yeast Pichia pastoris”, MicrobiologyOpen, vol. 10, 2021, : e1224.
Heß, M., Grollius, M., Duhay, V., Koopmeiners, S., Bloess, S., Fischer von Mollard, G.: Analysis of N-glycosylation in fungal l-amino acid oxidases expressed in the methylotrophic yeast Pichia pastoris. MicrobiologyOpen. 10, : e1224 (2021).
Heß, Marc, Grollius, Marvin, Duhay, Valentin, Koopmeiners, Simon, Bloess, Svenja, and Fischer von Mollard, Gabriele. “Analysis of N-glycosylation in fungal l-amino acid oxidases expressed in the methylotrophic yeast Pichia pastoris”. MicrobiologyOpen 10.4 (2021): e1224.

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