Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes.
Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes.
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DOI：
10.1128/mbio.02231-16
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发表时间：
2017-01-17
期刊：
影响因子：
6.4
通讯作者：
Glass NL
中科院分区：
文献类型：
--
作者：
Wu VW;Dana CM;Iavarone AT;Clark DS;Glass NL
将植物生物量分解为单糖对于生产第二代生物燃料和高价值生物产品至关重要。目前，丝状真菌产生的酶被用来将植物细胞壁多糖解构为可发酵的糖，用于生物精炼应用。在其中一些酶中观察到的翻译后N末端焦谷氨酸修饰发生在N末端谷氨酰胺或谷氨酸环化形成五元环时。这种修饰已经被证明对CBH-1和EG-1纤维素酶具有抗热变性的能力。在哺乳动物细胞中，谷氨酰环酶催化焦谷氨酸的形成。利用模式丝状真菌粗糙脉孢菌，我们鉴定了两个编码哺乳动物谷氨酰环酶同源蛋白的基因(qc-1和qc-2)。我们证明了QC-1和QC-2对于催化CBH-1和GH5-1上N-末端焦谷氨酸的形成是必不可少的。Δqc-1Δqc-2突变体产生的Cbh-1和Gh5-1缺乏N端焦谷氨酸修饰，对热变性更敏感，对Gh5-1对蛋白水解性切割更敏感。QC-1和QC-2是内质网(ER)定位蛋白。焦谷氨酸修饰预计会发生在许多具有不同功能的真菌蛋白中。真菌中谷氨酰环酶的鉴定可能对木质纤维素解酶的生产、异源表达以及围绕蛋白质稳定性的生物技术应用具有重要意义。焦谷氨酸修饰是将N-端谷氨酰胺或谷氨酸翻译后转化为环化氨基酸衍生物。这种修饰在动物系统中得到了很好的研究，但在真菌系统中的探索却很少。在粗糙脉孢菌中，我们发现这种修饰发生在内质网中，并由两种保守的酶催化，这两种酶在真菌王国中普遍保守。我们证明了这种修饰对CBH-1和GH5-1这两种用于工业植物细胞壁解构的重要纤维素酶的结构稳定性和对氨基肽酶的抗性是重要的。在粗毛拟青霉和其他丝状真菌的基因组中预测到的许多额外的真菌蛋白都被预测带有N端的焦谷氨酸修饰。焦谷氨酸的添加也可能是稳定分泌性蛋白质和多肽的一种有用的方法，这些蛋白质和多肽很容易在真菌生产系统中产生。
The breakdown of plant biomass to simple sugars is essential for the production of second-generation biofuels and high-value bioproducts. Currently, enzymes produced from filamentous fungi are used for deconstructing plant cell wall polysaccharides into fermentable sugars for biorefinery applications. A post-translational N-terminal pyroglutamate modification observed in some of these enzymes occurs when N-terminal glutamine or glutamate is cyclized to form a five-membered ring. This modification has been shown to confer resistance to thermal denaturation for CBH-1 and EG-1 cellulases. In mammalian cells, the formation of pyroglutamate is catalyzed by glutaminyl cyclases. Using the model filamentous fungus Neurospora crassa, we identified two genes (qc-1 and qc-2) that encode proteins homologous to mammalian glutaminyl cyclases. We show that qc-1 and qc-2 are essential for catalyzing the formation of an N-terminal pyroglutamate on CBH-1 and GH5-1. CBH-1 and GH5-1 produced in a Δqc-1 Δqc-2 mutant, and thus lacking the N-terminal pyroglutamate modification, showed greater sensitivity to thermal denaturation, and for GH5-1, susceptibility to proteolytic cleavage. QC-1 and QC-2 are endoplasmic reticulum (ER)-localized proteins. The pyroglutamate modification is predicted to occur in a number of additional fungal proteins that have diverse functions. The identification of glutaminyl cyclases in fungi may have implications for production of lignocellulolytic enzymes, heterologous expression, and biotechnological applications revolving around protein stability. Pyroglutamate modification is the post-translational conversion of N-terminal glutamine or glutamate into a cyclized amino acid derivative. This modification is well studied in animal systems but poorly explored in fungal systems. In Neurospora crassa, we show that this modification takes place in the ER and is catalyzed by two well-conserved enzymes, ubiquitously conserved throughout the fungal kingdom. We demonstrate that the modification is important for the structural stability and aminopeptidase resistance of CBH-1 and GH5-1, two important cellulase enzymes utilized in industrial plant cell wall deconstruction. Many additional fungal proteins predicted in the genome of N. crassa and other filamentous fungi are predicted to carry an N-terminal pyroglutamate modification. Pyroglutamate addition may also be a useful way to stabilize secreted proteins and peptides, which can be easily produced in fungal production systems.