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

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Analysis of N-glycosylation in fungal l-amino acid oxidases expressed in the methylotrophic yeast Pichia pastoris
MicrobiologyOpen ( IF 3.9 ) Pub Date : 2021-08-15 , DOI: 10.1002/mbo3.1224
Marc Christian Heß ₁ , Marvin Grollius ₁ , Valentin Duhay ₁ , Simon Koopmeiners ₁ , Svenja Bloess ₁ , Gabriele Fischer von Mollard ₁

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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.

中文翻译：

甲基营养酵母毕赤酵母中表达的真菌 L-氨基酸氧化酶的 N-糖基化分析

L-氨基酸氧化酶（LAAO）催化L-氨基酸氧化脱氨生成相应的α-酮酸。在这里，我们描述了四种真菌 LAAO 在毕赤酵母中的异源表达。来自胶孢炭疽菌的cg LAAO1 和来自粗糙脉孢菌的nc LAAO1 能够转化先前描述的来自真菌Hebeloma cylindrosporum的重组 9His -hc LAAO4 无法识别的底物，从而拓宽了潜在应用的底物谱。来自Fibroporia radiculosa的 9His- fr LAAO1 和来自Laccaria amethystine的 9His -la LAAO2 仅获得少量。所有四种酶均被 N-糖基化。我们生成了缺乏 N-糖基化位点的 9His- hc LAAO4 突变体，以进一步了解 N-糖基化的影响。所有四个预测的 N-糖基化位点均在P中表达的 9His- hc LAAO4 中糖基化。毕赤酵母。所有样品酸活化后，完全糖基化的 9His- hc LAAO4 和没有一个或所有 N-糖基化位点的变体的酶活性相似。然而，在没有 N-聚糖的变体中，未经酸处理的活性较低。这是由于天冬酰胺残基 N54 上缺乏高甘露糖基化的 N-聚糖所致。缺乏一种或所有其他 N-聚糖没有效果。我们的结果表明，采用更活跃的构象需要在生物合成过程中进行特定的 N-糖基化。

更新日期：2021-08-16

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