Department of Biotechnology, University of Verona, Verona, Italy.
Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Derio, Spain.
Proteins. 2023 Oct;91(10):1383-1393. doi: 10.1002/prot.26507. Epub 2023 May 10.
Cystathionine β-synthase (CBS) catalyzes the condensation of l-serine and l-homocysteine to give l-cystathionine in the transsulfuration pathway. Recently, a few O-acetylserine (l-OAS)-dependent CBSs (OCBSs) have been found in bacteria that can exclusively function with l-OAS. CBS from Toxoplasma gondii (TgCBS) can efficiently use both l-serine and l-OAS to form l-cystathionine. In this work, a series of site-specific variants substituting S84, Y160, and Y246 with hydrophobic residues found at the same positions in OCBSs was generated to explore the roles of the hydroxyl moieties of these residues as determinants of l-serine/l-OAS preference in TgCBS. We found that the S84A/Y160F/Y246V triple mutant behaved like an OCBS in terms of both substrate requirements, showing β-replacement activity only with l-OAS, and pH optimum, which is decreased by ~1 pH unit. Formation of a stable aminoacrylate upon reaction with l-serine is prevented by the triple mutation, indicating the importance of the H-bonds between the hydroxyl groups of Y160, Y246, and S84 with l-serine in formation of the intermediate. Analysis of the independent effect of each mutation on TgCBS activity and investigation of the protein-aminoacrylate complex structure allowed for the conclusion that the hydroxyl group of Y246 has a major, but not exclusive, role in controlling the l-serine preference by efficiently stabilizing its leaving group. These studies demonstrate that differences in substrate specificity of CBSs are controlled by natural variations in as few as three residue positions. A better understanding of substrate specificity in TgCBS will facilitate the design of new antimicrobial compounds.
胱硫醚β-合酶（CBS）在转硫途径中催化 l-丝氨酸和 l-同型半胱氨酸缩合生成 l-胱硫醚。最近，在细菌中发现了几种 O-乙酰丝氨酸（l-OAS）依赖性 CBS（OCBS），它们只能与 l-OAS 一起发挥作用。刚地弓形虫（TgCBS）的 CBS 可以有效地利用 l-丝氨酸和 l-OAS 形成 l-胱硫醚。在这项工作中，生成了一系列取代 S84、Y160 和 Y246 的定点突变体，这些取代的氨基酸是在 OCBS 中发现的同源疏水残基，用于探索这些残基的羟基作为 TgCBS 中 l-丝氨酸/l-OAS 偏好决定因素的作用。我们发现，S84A/Y160F/Y246V 三重突变体在底物要求方面表现出 OCBS 的特征，仅与 l-OAS 表现出β-取代活性，并且 pH 最适值降低了约 1 pH 单位。三重突变阻止了与 l-丝氨酸反应形成稳定的氨基丙烯酸酯，表明 Y160、Y246 和 S84 的羟基与 l-丝氨酸之间的氢键在形成中间体过程中的重要性。对每个突变对 TgCBS 活性的独立影响的分析以及对蛋白-氨基丙烯酸酯复合物结构的研究表明，Y246 的羟基在控制 l-丝氨酸偏好方面起着主要作用，但不是唯一作用，它可以有效地稳定离去基团。这些研究表明，CBS 底物特异性的差异是由少数三个残基位置的自然变异控制的。更好地了解 TgCBS 的底物特异性将有助于设计新的抗菌化合物。
