用于化学酶法合成 K48/K63 异型和支化泛素链的生物相容性赖氨酸保护基,ACS Central Science

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用于化学酶法合成 K48/K63 异型和支化泛素链的生物相容性赖氨酸保护基
ACS Central Science ( IF 12.7 ) Pub Date : 2023-07-15 , DOI: 10.1021/acscentsci.3c00389
Toshiki Mikami ₁ , Sohei Majima ₁ , Haewon Song ₁ , Jeffrey W Bode ₁

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阐明异型和支化泛素 (Ub) 链的新兴生物学功能需要新的策略来制备具有确定长度和连接性的它们。虽然使用表达的 E1 激活酶和 E2 结合酶进行体外酶组装可以提供同型链，但支链的合成通常需要赖氨酸的广泛突变或其他序列修饰。K48-和K63偏向的E2-结合酶和两个新的氨基甲酸酯保护基的组合─吡哆醛5′-磷酸(PLP)-可裂解的氨基丁酰胺氨基甲酸酯( Abac组)和高碘酸盐可裂解的氨基丁酸o l氨基甲酸酯（Aboc 基团）─提供异型和支链 Ub 三聚体、四聚体和五聚体的合成策略。Abac 和 Aboc 保护的赖氨酸很容易制备并掺入合成泛素单体中。由于这些掩蔽基团含有碱性胺，它们保留了 Ub 结构的整体电荷和特性，促进折叠和酶促缀合。_{这些保护基团可以通过 PLP 或 NaIO 4}的缓冲溶液化学选择性地从折叠的 Ub 链和单体中去除。通过在 Ub 受体上掺入可裂解的 C 端 His 标签，链构建、迭代 Abac 脱保护和全局 Aboc 裂解的整个过程可以在树脂支持物上进行，从而无需处理和纯化中间体低聚物。Ub 单体的简单调节提供了各种 K48/K63 支链，包括以前无法通过合成或生化方法获得的四聚体和五聚体。

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Biocompatible Lysine Protecting Groups for the Chemoenzymatic Synthesis of K48/K63 Heterotypic and Branched Ubiquitin Chains

The elucidation of emerging biological functions of heterotypic and branched ubiquitin (Ub) chains requires new strategies for their preparation with defined lengths and connectivity. While in vitro enzymatic assembly using expressed E1-activating and E2-conjugating enzymes can deliver homotypic chains, the synthesis of branched chains typically requires extensive mutations of lysines or other sequence modifications. The combination of K48- and K63-biased E2-conjugating enzymes and two new carbamate protecting groups─pyridoxal 5′-phosphate (PLP)-cleavable aminobutanamide carbamate (Abac group) and periodate-cleavable aminobutanol carbamate (Aboc group)─provides a strategy for the synthesis of heterotypic and branched Ub trimers, tetramers, and pentamers. The Abac- and Aboc-protected lysines are readily prepared and incorporated into synthetic ubiquitin monomers. As these masking groups contain a basic amine, they preserve the overall charge and properties of the Ub structure, facilitating folding and enzymatic conjugations. These protecting groups can be chemoselectively removed from folded Ub chains and monomers by buffered solutions of PLP or NaIO₄. Through the incorporation of a cleavable C-terminal His-tag on the Ub acceptor, the entire process of chain building, iterative Abac deprotections, and global Aboc cleavage can be conducted on a resin support, obviating the need for handling and purification of the intermediate oligomers. Simple modulation of the Ub monomers affords various K48/K63 branched chains, including tetramers and pentamers not previously accessible by synthetic or biochemical methods.

更新日期：2023-07-15

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