Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), Ås, Norway.
Department of Biotechnology and Food Science, NOBIPOL, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.
Protein Sci. 2018 Sep;27(9):1636-1650. doi: 10.1002/pro.3451.
The catalytically crucial N-terminal histidine (His1) of fungal lytic polysaccharide monooxygenases (LPMOs) is post-translationally modified to carry a methylation. The functional role of this methylation remains unknown. We have carried out an in-depth functional comparison of two variants of a family AA9 LPMO from Thermoascus aurantiacus (TaLPMO9A), one with, and one without the methylation on His1. Various activity assays showed that the two enzyme variants are identical in terms of substrate preferences, cleavage specificities and the ability to activate molecular oxygen. During the course of this work, new functional features of TaLPMO9A were discovered, in particular the ability to cleave xyloglucan, and these features were identical for both variants. Using a variety of techniques, we further found that methylation has minimal effects on the pK of His1, the affinity for copper and the redox potential of bound copper. The two LPMOs did, however, show clear differences in their resistance against oxidative damage. Studies with added hydrogen peroxide confirmed recent claims that low concentrations of H O boost LPMO activity, whereas excess H O leads to LPMO inactivation. The methylated variant of TaLPMO9A, produced in Aspergillus oryzae, was more resistant to excess H O and showed better process performance when using conditions that promote generation of reactive-oxygen species. LPMOs need to protect themselves from reactive oxygen species generated in their active sites and this study shows that methylation of the fully conserved N-terminal histidine provides such protection.
真菌溶细胞多糖单加氧酶（LPMOs）的催化关键的 N 端组氨酸（His1）经翻译后修饰，携带一个甲基化。该甲基化的功能作用仍然未知。我们对来自嗜热子囊菌（Thermoascus aurantiacus）的 AA9 家族 LPMO 的两种变体（一种带有，一种不带 His1 上的甲基化）进行了深入的功能比较。各种活性测定表明，两种酶变体在底物偏好、切割特异性和激活分子氧的能力方面完全相同。在这项工作的过程中，发现了 TaLPMO9A 的新功能特征，特别是能够切割木葡聚糖，并且这两种变体都具有这些特征。使用多种技术，我们进一步发现甲基化对 His1 的 pK、铜亲和力和结合铜的氧化还原电位的影响最小。然而，这两种 LPMO 在其对氧化损伤的抵抗力方面表现出明显的差异。添加过氧化氢的研究证实了最近的说法，即低浓度的 H2O2 可以提高 LPMO 的活性，而过量的 H2O2 会导致 LPMO 失活。在米曲霉中产生的 TaLPMO9A 的甲基化变体对过量的 H2O2 具有更强的抵抗力，并且在使用促进活性氧产生的条件下显示出更好的处理性能。LPMO 需要保护其活性位点中产生的活性氧，本研究表明，完全保守的 N 端组氨酸的甲基化提供了这种保护。
