南方科技大学陶丽芝团队报道了自由基SAM酶PylB利用SAM作为辅助因子，在热解赖氨酸的生物合成中产生赖氨酸自由基中间体。相关研究成果于2024年3月1日发表在国际顶尖学术期刊《美国化学会杂志》。

吡咯赖氨酸是由天然遗传密码编码的第22个氨基酸，是产甲烷古菌将甲胺分解代谢为甲烷所必需的。吡咯赖氨酸的结构由甲基化吡咯羧酸盐组成，该羧酸盐通过酰胺键与l-赖氨酸中的ε-氨基相连。吡咯赖氨酸的生物合成需要三种酶：PylB、PylC和PylD。PylB是一种自由基S-腺苷-1-甲硫氨酸（SAM）酶，催化第一个生物合成步骤，即l-赖氨酸异构化为甲基鸟氨酸。

PylC催化甲基鸟氨酸和第二个l-赖氨酸的ATP依赖性连接形成l-赖氨素-Nε-甲基鸟氨酸。最后一个生物合成步骤由PylD催化，通过PylC产物的氧化形成吡咯赖氨酸。虽然PylC和PylD的酶促反应已经通过X射线晶体学和体外研究得到了很好的表征，但对PylB的机制理解仍然相对有限。

该文中，研究人员报道了PylB通过l-赖氨酸异构化形成甲基鸟氨酸的首次体外活性。研究人员还鉴定了一种被捕获的赖氨酰C4自由基中间体，其电子结构和几何结构通过EPR和ENDOR光谱得到了很好的表征。此外，证明SAM在PylB催化中起催化辅因子的作用，而不是典型的共底物。

该项工作为阐明PylB在吡咯赖氨酸生物合成过程中，催化的碳骨架重排反应提供了详细的机制证据。

附：英文原文

Title: Radical SAM Enzyme PylB Generates a Lysyl Radical Intermediate in the Biosynthesis of Pyrrolysine by Using SAM as a Cofactor

Author: Baixu Ma, R. David Britt, Lizhi Tao

Issue&Volume: March 1, 2024

Abstract: Pyrrolysine, the 22nd amino acid encoded by the natural genetic code, is essential for methanogenic archaea to catabolize methylamines into methane. The structure of pyrrolysine consists of a methylated pyrroline carboxylate that is linked to the ε-amino group of the l-lysine via an amide bond. The biosynthesis of pyrrolysine requires three enzymes: PylB, PylC, and PylD. PylB is a radical S-adenosyl-l-methionine (SAM) enzyme and catalyzes the first biosynthetic step, the isomerization of l-lysine into methylornithine. PylC catalyzes an ATP-dependent ligation of methylornithine and a second l-lysine to form l-lysine-Nε-methylornithine. The last biosynthetic step is catalyzed by PylD via oxidation of the PylC product to form pyrrolysine. While enzymatic reactions of PylC and PylD have been well characterized by X-ray crystallography and in vitro studies, mechanistic understanding of PylB is still relatively limited. Here, we report the first in vitro activity of PylB to form methylornithine via the isomerization of l-lysine. We also identify a lysyl C4 radical intermediate that is trapped, with its electronic structure and geometric structure well characterized by EPR and ENDOR spectroscopy. In addition, we demonstrate that SAM functions as a catalytic cofactor in PylB catalysis rather than canonically as a cosubstrate. This work provides detailed mechanistic evidence for elucidating the carbon backbone rearrangement reaction catalyzed by PylB during the biosynthesis of pyrrolysine.

DOI: 10.1021/jacs.3c11266

Source: https://pubs.acs.org/doi/abs/10.1021/jacs.3c11266