Centre for Protein Science and Crystallography, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China.
Li Dak Sum Yip Yio Chin R & D Centre for Chinese Medicine, The Chinese University of Hong Kong, Hong Kong, China.
Int J Mol Sci. 2021 Oct 27;22(21):11598. doi: 10.3390/ijms222111598.
Ribosome-inactivating proteins (RIPs) hydrolyze the N-glycosidic bond and depurinate a specific adenine residue (A-4324 in rat 28S ribosomal RNA, rRNA) in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. In this study, we have purified and characterized lyophyllin, an unconventional RIP from , an edible mushroom. The protein resembles peptidase M35 domain of peptidyl-Lys metalloendopeptidases. Nevertheless, protein either from the mushroom or in recombinant form possessed N-glycosidase and protein synthesis inhibitory activities. A homology model of lyophyllin was constructed. It was found that the zinc binding pocket of this protein resembles the catalytic cleft of a classical RIP, with key amino acids that interact with the adenine substrate in the appropriate positions. Mutational studies showed that E122 may play a role in stabilizing the positively charged oxocarbenium ion and H121 for protonating N-3 of adenine. The tyrosine residues Y137 and Y104 may be used for stacking the target adenine ring. This work first shows a protein in the peptidase M35 superfamily based on conserved domain search possessing N-glycosidase activity.
核糖体失活蛋白（RIPs）可水解 N-糖苷键，并使 rRNA 上保守的 α-桑辛/蓖麻毒素环（α-SRL）中的特定腺嘌呤残基（大鼠 28S rRNA 中的 A-4324）脱嘌呤。在这项研究中，我们从一种可食用蘑菇中纯化并鉴定了 lyophyllin，这是一种非典型的 RIP。该蛋白类似于肽酰-Lys 金属内肽酶的肽酶 M35 结构域。然而，无论是来自蘑菇的蛋白质还是重组形式的蛋白质都具有 N-糖苷酶和蛋白质合成抑制活性。构建了 lyophyllin 的同源模型。发现该蛋白的锌结合口袋类似于经典 RIP 的催化裂缝，关键氨基酸与适当位置的腺嘌呤底物相互作用。突变研究表明，E122 可能在稳定带正电荷的氧碳翁离子和 H121 质子化腺嘌呤的 N-3 方面发挥作用。酪氨酸残基 Y137 和 Y104 可能用于堆积靶腺嘌呤环。这项工作首次表明，基于保守结构域搜索，具有 N-糖苷酶活性的肽酶 M35 超家族中的一种蛋白。
