Analysis of N-glycosylation in fungal l-amino acid oxidases expressed in the methylotrophic yeast Pichia pastoris.
Pichia pastoris
fungi
glycosylation
heterologous expression
l-amino acid oxidases
Journal
MicrobiologyOpen
ISSN: 2045-8827
Titre abrégé: Microbiologyopen
Pays: England
ID NLM: 101588314
Informations de publication
Date de publication:
08 2021
08 2021
Historique:
revised:
06
07
2021
received:
14
04
2021
accepted:
08
07
2021
entrez:
30
8
2021
pubmed:
31
8
2021
medline:
6
1
2022
Statut:
ppublish
Résumé
l-amino acid oxidases (LAAOs) catalyze the oxidative deamination of l-amino acids to corresponding α-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.
Identifiants
pubmed: 34459552
doi: 10.1002/mbo3.1224
pmc: PMC8364938
doi:
Substances chimiques
L-Amino Acid Oxidase
EC 1.4.3.2
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e1224Informations de copyright
© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
Références
J Exp Biol. 2005 Sep;208(Pt 18):3609-22
pubmed: 16155232
Appl Microbiol Biotechnol. 2017 Apr;101(7):2853-2864
pubmed: 27986991
J Biosci Bioeng. 2007 Sep;104(3):218-23
pubmed: 17964487
Biochem J. 1950 Feb;46(2):210-9
pubmed: 16748663
Planta. 1992 Aug;188(1):13-8
pubmed: 24178194
Appl Microbiol Biotechnol. 2019 Mar;103(5):2229-2241
pubmed: 30631897
J Biol Chem. 2002 May 24;277(21):19080-6
pubmed: 11907037
Protein Expr Purif. 2007 Mar;52(1):89-95
pubmed: 17127077
Biotechnology (N Y). 1994 Feb;12(2):181-4
pubmed: 7764433
Front Microbiol. 2020 May 19;11:977
pubmed: 32508788
Biotechniques. 2004 Jan;36(1):152-4
pubmed: 14740498
Toxicon. 2014 Oct;89:32-44
pubmed: 25009089
FEBS J. 2010 Jan;277(2):453-65
pubmed: 20015076
EMBO J. 2000 Aug 15;19(16):4204-15
pubmed: 10944103
J Agric Food Chem. 2011 May 11;59(9):4519-26
pubmed: 21456553
Bioorg Med Chem. 2006 Oct 15;14(20):7034-43
pubmed: 16809041
Arch Pharm Res. 2000 Oct;23(5):477-81
pubmed: 11059827
Microbiologyopen. 2020 Oct;9(10):e1112
pubmed: 32852125
Biol Bull. 2018 Aug;235(1):52-61
pubmed: 30160994
Biochimie. 2009 Apr;91(4):490-501
pubmed: 19135502
J Agric Food Chem. 2011 Sep 14;59(17):9142-9
pubmed: 21797276
Molecules. 2017 Dec 20;22(12):
pubmed: 29261108
Biotechnol Appl Biochem. 1999 Dec;30(3):193-200
pubmed: 10574687
Mycol Res. 2008 Dec;112(Pt 12):1453-64
pubmed: 18675352
Appl Microbiol Biotechnol. 2013 Nov;97(21):9323-41
pubmed: 24077723
Appl Biochem Biotechnol. 2012 May;167(1):1-13
pubmed: 22367642
Bioorg Med Chem. 2004 Jun 1;12(11):2881-6
pubmed: 15142548
Biomed Res Int. 2014;2014:196754
pubmed: 24738050
J Biol Chem. 1990 Oct 5;265(28):17246-51
pubmed: 2145270