Molecular insight into the enzymatic macrocyclization of multiply backbone N-methylated peptides
Molecular insight into the enzymatic macrocyclization of multiply backbone N-methylated peptides
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DOI：
10.1101/2022.07.21.500988
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发表时间：
2023-04
期刊：
影响因子：
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通讯作者：
Emmanuel Matabaro;Haigang Song;Fabio Gherlone;L. Sonderegger;Andrew M. Giltrap;Samuel Liver;A. Gossert;M. Künzler;J. Naismith
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作者：
Emmanuel Matabaro;Haigang Song;Fabio Gherlone;L. Sonderegger;Andrew M. Giltrap;Samuel Liver;A. Gossert;M. Künzler;J. Naismith
OphP酶是生物合成大环肽Omepristin A所必需的，Omepristin A是由木材降解真菌Omepristus olearius产生的具有9个骨架N-甲基化的十二聚体。OphP和肽-前体蛋白OphMA在酵母中的异源表达产生了Ompectin A。因此，假设Oph P具有双重功能;催化OphMA中肽中间体的内切蛋白水解释放，以及多重α-N-甲基化核心肽的大环化，同时释放C-末端跟随肽。在我们的体外活性测定中，OphP对α-N-甲基化肽显示出强的内切蛋白水解和大环化酶活性，但不能切割OphMA。该酶对疏水的、高度α-N-甲基化的肽和P1位点的α-N-甲基化甘氨酸残基具有强烈的偏好性。OphP采用典型的脯氨酰寡肽酶（POP）折叠，主要是疏水性底物结合裂缝，和一个小的疏水性P1结合口袋。我们证明，OphP是一种POP型大环化酶的特异性和底物途径的活性位点不同于其他成员的家庭。这些结果可以利用生物技术生产的大环肽与多个骨干N-甲基化，这是有趣的，由于其有利的药理学性质。
The enzyme OphP is essential for the biosynthesis of the macrocyclic peptide omphalotin A, a dodecamer with 9 backbone N-methylations produced by the wood-degrading fungus Omphalotus olearius. Heterologous expression of OphP and the peptide-precursor protein OphMA in yeast, yields omphalotin A. Thus, Oph P was hypothesized to have a dual function; catalyzing both endoproteolytic release of a peptide intermediate from OphMA, and macrocyclization of the multiply α-N-methylated core peptide with concomitant release of a C-terminal follower peptide. In our in vitro activity assays, OphP showed robust endoproteolytic and macrocyclase activity on α-N-methylated peptides but was unable to cleave OphMA. The enzyme had a strong preference for hydrophobic, highly α-N-methylated peptides and an α-N-methylated glycine residue at the P1 site. OphP adopts a canonical prolyl oligopeptidase (POP) fold with a predominantly hydrophobic substrate binding cleft, and a small and hydrophobic P1 binding pocket. We demonstrate that OphP is a POP-type macrocyclase with a specificity and a substrate route to the active site different from other members of the family. These results could be exploited for the biotechnological production of macrocyclic peptides with multiple backbone N-methylations, which are interesting due to their favorable pharmacological properties.