Structural, Kinetic, and Mechanistic Analysis of an Asymmetric 4-Oxalocrotonate Tautomerase Trimer
Structural, Kinetic, and Mechanistic Analysis of an Asymmetric 4-Oxalocrotonate Tautomerase Trimer
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DOI：
10.1021/acs.biochem.9b00303
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发表时间：
2019-06-04
期刊：
影响因子：
2.9
通讯作者：
Whitman, Christian P.
中科院分区：
文献类型：
--
作者：
Baas, Bert-Jan;Medellin, Brenda P.;Whitman, Christian P.
从宽叶伯克霍尔德氏菌中分离出一个4-草酰基互变三聚体(4-OT)，并进行了动力学、机理和结构分析。该酶是所描述的第三种4-OT低聚体状态，与同六聚体和异六聚体一起。4-OT三聚体是互变清除酶超家族(TSF)4-OT亚群内的一小部分序列(133个序列)的一部分。TSF有两个不同的特征：成员由一个单一的β-α-β单位(同六聚体和异六聚体)或两个连续连接的β-α-β单位(三聚体)组成，通常具有一个催化的氨基末端脯氨酸。该酶被命名为融合的4-OT，其功能是4-OT，其中活性部位组(Pro-1、Arg-39、Arg-76、Phe-115、Arg-127)与恶臭假单胞菌Mt-2的典型4-OT中的活性部位群相同。2-氧代-3-戊烯酸的失活表明融合的4-OT的Pro-1具有低的pK(A)，使Pro-1能够作为普通碱发挥作用。融合的4-OT的一个显著特征是结构中没有P3旋转对称(1.5埃分辨率)。三聚体的不对称排列并不是由于两个β-α-β构建块的融合，因为一个破坏两个单元之间的共价键(以产生异六聚体)的工程“未融合”变体呈现相同的不对称齐聚状态。目前尚不清楚不同的活性中心配置如何对观察到的总体活动做出贡献，以及这种不对称性是否在TSF成员的进化中具有生物学目的或作用。
A 4-oxalocrotonate tautomerase (4-OT) trimer has been isolated from Burkholderia lata, and a kinetic, mechanistic, and structural analysis has been performed. The enzyme is the third described oligomer state for 4-OT along with a homo- and heterohexamer. The 4-OT trimer is part of a small subset of sequences (133 sequences) within the 4-OT subgroup of the tautomerase superfamily (TSF). The TSF has two distinct features: members are composed of a single beta-alpha-beta unit (homo- and heterohexamer) or two consecutively joined beta-alpha-beta units (trimer) and generally have a catalytic amino-terminal proline. The enzyme, designated as fused 4-OT, functions as a 4-OT where the active site groups (Pro-1, Arg-39, Arg-76, Phe-115, Arg-127) mirror those in the canonical 4-OT from Pseudomonas putida mt-2. Inactivation by 2-oxo-3-pentynoate suggests that Pro-1 of fused 4-OT has a low pK(a) enabling the prolyl nitrogen to function as a general base. A remarkable feature of the fused 4-OT is the absence of P3 rotational symmetry in the structure (1.5 angstrom resolution). The asymmetric arrangement of the trimer is not due to the fusion of the two beta-alpha-beta building blocks because an engineered "unfused" variant that breaks the covalent bond between the two units (to generate a heterohexamer) assumes the same asymmetric oligomerization state. It remains unknown how the different active site configurations contribute to the observed overall activities and whether the asymmetry has a biological purpose or role in the evolution of TSF members.