Mechanistic Insights into DyPB from Rhodococcus jostii RHA1 Via Kinetic Characterization
Mechanistic Insights into DyPB from Rhodococcus jostii RHA1 Via Kinetic Characterization
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通过动力学表征从红球菌 jostii RHA1 中了解 DyPB 的机制
DOI：
10.1021/acscatal.1c00703
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发表时间：
2021
期刊：
影响因子：
12.9
通讯作者：
Li, Ping
中科院分区：
文献类型：
--
作者：
Shrestha, Ruben;Jia, Kaimin;Khadka, Samiksha;Eltis, Lindsay D.;Li, Ping
染料脱色过氧化物酶（DyPs）是近年来发现的一类具有生物工程潜力的血红素过氧化物酶。然而，远端天冬氨酸和精氨酸在催化中的作用仍有待研究。在这里，我们阐明了他们的作用DyPB从Rhodococcus jostiiRHA 1使用稳态和瞬态动力学，pH速率关系，粘度和溶剂动力学同位素效应（sKIE）。与以前的数据一致，取代的远端天冬氨酸降低化合物（Cpd）I形成的速率远低于取代的远端精氨酸。结构数据的检查表明，这可能是因为远端天冬氨酸比其他B类DyP更远离DyPB中的溶剂配体。用2，2 ′-连氮基-双（3-乙基苯并噻唑啉-6-磺酸）（ABTS）替代远端天冬氨酸使Cpd I还原速率降低了1.11倍。观察到的WT（kcat/KM）ABTS的逆KIE为0.13，与催化循环期间从血红素铁解离的三个沃茨一致，表明在DyP催化的反应中quorelase的机械重要性。Cpd I还原沿着的inversesKIE为0.25，与其他观察结果一起表明Cpd I可能处于“湿”形式，并可能通过未检测到的Cpd II进行两次连续的1 e-还原。的粘度效应，sKIE，和pH-速率曲线的化合物I形成表明，虽然化合物0去质子化可能是限速，O-O断裂发挥更重要的作用，在DyPB比其他B类酶。有人进一步提出，限速构象变化之前的步骤，返回酶的静息状态ABTS氧化。通过强调类似DyPs之间的机制差异，这项研究提供了对一类重要酶的深入了解。
Dye-decolorizing peroxidases (DyPs) comprise a recently discovered family of heme peroxidases with considerable biotechnological potential. However, the roles of the distal aspartate and arginine in catalysis remain underexplored. Here, we elucidated their roles in DyPB fromRhodococcus jostiiRHA1 using steady- and transient-state kinetics, pH–rate relationship, viscosity, and solvent kinetic isotope effect (sKIE). Consistent with previous data, substitution of the distal aspartate reduced the rate of compound (Cpd) I formation much less dramatically than substitution of the distal arginine. Inspection of the structural data suggests that this may be because the distal aspartate is much farther away from the solvent ligand in DyPB than in other B-class DyPs. Replacement of the distal aspartate decreased the rate of Cpd I reduction with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) by ∼11-fold. An observed inversesKIE of 0.13 for the WT (kcat/KM)ABTSis consistent with three waters dissociating from the heme iron during a catalytic cycle, indicating mechanistic importance ofaquorelease in DyP-catalyzed reactions. An inversesKIE of 0.25 for Cpd I reduction along with other observations implied that Cpd I is likely in a “wet” form and may undergo two successive 1e–-reductions via an undetected Cpd II. The viscosity effect,sKIE, and pH–rate profiles of Cpd I formation demonstrated that, while Cpd 0 deprotonation is likely rate-limiting, O–O scission plays a more significant role in DyPB than in other B-class enzymes. It is further proposed that a rate-limiting conformational change precedes the step that returns the enzyme to its resting state in ABTS oxidation. By highlighting mechanistic differences between similar DyPs, this study provides insight into an important class of enzymes.
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