A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes.
A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes.
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DOI：
10.1128/aem.01768-16
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发表时间：
2016-11-15
影响因子：
4.4
通讯作者：
Yoshida M
中科院分区：
文献类型：
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作者：
Kojima Y;Várnai A;Ishida T;Sunagawa N;Petrovic DM;Igarashi K;Jellison J;Goodell B;Alfredsen G;Westereng B;Eijsink VG;Yoshida M
真菌分泌一系列糖苷水解酶和溶解性多糖单加氧酶（LPMO）降解植物多糖。褐腐真菌，如密粘褶菌，往往有很少的LPMO，这些酶的信息是稀缺的。G. trabeum编码4个辅助活性9（AA 9）LPMO（GtLPMO 9 s），其编码序列是从cDNA中扩增的。由于选择性剪接，GtLPMO 9A的两种变体似乎产生，单结构域变体GtLPMO 9A-1和更长的变体GtLPMO 9A-2，其含有包含约55个残基的C-末端结构域，没有预测的功能。我们已经在毕赤酵母中过表达了遗传学上不同的GtLPMO 9A-2，并研究了其性质。使用高效阴离子交换色谱-脉冲安培检测（HPAEC-PAD）和质谱（MS）进行的标准分析表明，GtLPMO 9A-2对纤维素、羧甲基纤维素和木葡聚糖具有活性。重要的是，与其他已知的木葡聚糖活性LPMO相比，GtLPMO 9 A-2具有广泛的特异性，在沿着木葡聚糖的β-葡聚糖主链的任何位置沿着裂解，而不管取代。使用动态粘度测量来比较GtLPMO 9A-2与充分表征的半纤维素分解LPMO的半纤维素分解作用，来自粗糙脉孢菌的NcLPMO 9 C揭示GtLPMO 9A-2在解聚木葡聚糖方面更有效。这些测量还揭示了对葡甘露聚糖的较小活性，其不能通过HPAEC-PAD和MS分析可溶性产物来检测，并且低于NcLPM 0 9 C的活性。用共聚底物的实验显示半纤维素涂层对纤维素分解LPMO活性的抑制作用，并且没有揭示GtLPMO 9A-2的额外活性。这些结果提供了对G. trabeum，并提供了一种新的灵敏的方法，动态粘度的测量，用于监测LPMO活性。重要性目前，只有少数方法可用于分析溶解性多糖单加氧酶（LPMO）活性的终产物，最常见的是液相色谱法和质谱法。在这里，我们提出了一种替代的和敏感的方法的基础上测量的动态粘度实时连续监测LPMO活性的水溶性半纤维素，如木葡聚糖的存在下。我们已经使用这些新的和现有的分析方法来表征木葡聚糖活性LPMO从褐腐真菌。这种酶，GtLPMO 9A-2，不同于先前表征的LPMO，具有广泛的底物特异性，能够几乎随机地切割木葡聚糖主链。GtLPM 09 A-2优先作用于游离木葡聚糖，表明优先选择将纤维素纤维拴在一起的木葡聚糖链。GtLPMO 9A-2的木葡聚糖降解潜力表明，在褐腐的初始阶段，通过降解初生细胞壁降低木材强度。
Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity. IMPORTANCE Currently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown-rot fungus. This enzyme, GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown rot through degradation of the primary cell wall.