Wu Vincent W, Dana Craig M, Iavarone Anthony T, Clark Douglas S, Glass N Louise
Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, USA.
Energy Biosciences Institute, University of California, Berkeley, Berkeley, California, USA.
mBio. 2017 Jan 17;8(1):e02231-16. doi: 10.1128/mBio.02231-16.
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.
将植物生物质分解为单糖对于第二代生物燃料和高价值生物产品的生产至关重要。目前，丝状真菌产生的酶用于将植物细胞壁多糖解构为可发酵糖，用于生物精炼应用。在其中一些酶中观察到的翻译后N端焦谷氨酸修饰，发生在N端谷氨酰胺或谷氨酸环化形成五元环时。已证明这种修饰赋予CBH - 1和EG - 1纤维素酶抗热变性能力。在哺乳动物细胞中，焦谷氨酸的形成由谷氨酰胺环化酶催化。利用丝状真菌粗糙脉孢菌作为模型，我们鉴定出两个基因（qc - 1和qc - 2），它们编码与哺乳动物谷氨酰胺环化酶同源的蛋白质。我们表明qc - 1和qc - 2对于催化CBH - 1和GH5 - 1上N端焦谷氨酸的形成至关重要。在Δqc - 1Δqc - 2突变体中产生的CBH - 1和GH5 - 1，因此缺乏N端焦谷氨酸修饰，对热变性表现出更高的敏感性，对于GH5 - 1，还表现出对蛋白水解切割的敏感性。QC - 1和QC - 2是内质网（ER）定位的蛋白质。预计焦谷氨酸修饰会发生在许多具有不同功能的其他真菌蛋白质中。真菌中谷氨酰胺环化酶的鉴定可能对木质纤维素分解酶的生产、异源表达以及围绕蛋白质稳定性的生物技术应用产生影响。
焦谷氨酸修饰是N端谷氨酰胺或谷氨酸向环化氨基酸衍生物的翻译后转化。这种修饰在动物系统中已得到充分研究，但在真菌系统中研究较少。在粗糙脉孢菌中，我们表明这种修饰发生在内质网中，由两种在整个真菌界普遍保守的保守酶催化。我们证明这种修饰对于CBH - 1和GH5 - 1的结构稳定性和氨肽酶抗性很重要，这两种是工业植物细胞壁解构中使用的重要纤维素酶。在粗糙脉孢菌和其他丝状真菌的基因组中预测的许多其他真菌蛋白质预计携带N端焦谷氨酸修饰。添加焦谷氨酸也可能是稳定分泌蛋白和肽的有用方法，这些蛋白和肽可以在真菌生产系统中轻松产生。
