Department of Chemistry, The University of British Columbia, Okanagan Campus, 3247 University Way, Kelowna, British Columbia V1V1V7, Canada.
Biochemistry. 2020 Nov 10;59(44):4250-4261. doi: 10.1021/acs.biochem.0c00683. Epub 2020 Oct 28.
Lanthionine synthase from the oral bacterium is a fold type II pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the β-replacement of l-cysteine by a second molecule of l-cysteine to form HS and l-lanthionine. The -isomer of the latter product is incorporated into the peptidoglycan layer. Herein, we investigated the catalytic role of S224, which engages in hydrogen-bond contact with the terminal carboxylate of l-lanthionine in the closed conformation of the enzyme. Unexpectedly, the S224A variant elicited a 7-fold increase in the turnover rate for HS and lanthionine formation and a 70-fold faster rate constant for the formation of the α-aminoacrylate intermediate compared to the wild-type enzyme. Presteady state kinetic analysis further showed that the reaction between S224A and l-cysteine leads to the formation of the more reactive ketoenamine tautomer of the α-aminoacrylate. The α-aminoacrylate with the protonated Schiff base is not an observable intermediate in the analogous reaction with the wild type, which may account for its attenuated kinetic properties. However, the S224A substitution is detrimental to other aspects of the catalytic cycle; it facilitates the α,β-elimination of l-lanthionine, and it weakens the enzyme's catalytic preference for the formation of l-lanthionine over that of l-cystathionine.
口腔细菌中的高丝氨酸内酯合成酶是一种折叠类型 II 的吡哆醛-5'-磷酸（PLP）依赖性酶，它催化第二个 l-半胱氨酸分子对 l-半胱氨酸的β取代，形成 HS 和 l-高丝氨酸内酯。后者产物的 -异构体被掺入肽聚糖层中。在此，我们研究了 S224 的催化作用，S224 与酶的封闭构象中 l-高丝氨酸内酯的末端羧酸盐进行氢键接触。出乎意料的是，与野生型酶相比，S224A 变体使 HS 和高丝氨酸内酯形成的周转速率提高了 7 倍，α-氨基丙烯酸中间物形成的速率常数提高了 70 倍。预稳态动力学分析进一步表明，S224A 和 l-半胱氨酸之间的反应导致形成更具反应性的α-氨基丙烯酸酮烯胺互变异构体。与野生型类似反应中未观察到带质子的 Schiff 碱的α-氨基丙烯酸中间物，这可能是其减弱的动力学特性的原因。然而，S224A 取代不利于催化循环的其他方面；它促进了 l-高丝氨酸内酯的α,β-消除，并且削弱了酶对 l-高丝氨酸内酯形成的催化偏好，而不是对 l-胱硫醚的形成。
