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首页> 外文期刊>Journal of Molecular Biology >Nylon-oligomer Degrading Enzyme/Substrate Complex: Catalytic Mechanism of 6-Aminohexanoate-dimer Hydrolase.
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Nylon-oligomer Degrading Enzyme/Substrate Complex: Catalytic Mechanism of 6-Aminohexanoate-dimer Hydrolase.

机译：尼龙-低聚物降解酶/底物复合物：6-氨基己酸二聚体水解酶的催化机理。

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摘要

We performed X-ray crystallographic analyses of 6-aminohexanoate-dimer hydrolase (Hyb-24DN), an enzyme responsible for the degradation of nylon-6, an industry by-product, and of a complex between Hyb-24DN-A(112) (S112A-mutant of Hyb-24DN) and 6-aminohexanoate-linear dimer (Ald) at 1.58 A and 1.4 A resolution, respectively. In Hyb-24DN, Asp181-O(delta) forms hydrogen bonds with Tyr170-O(eta), -two of the catalytic and binding amino acids, and a loop between Asn167 and Val177. This state is the so-called open form, allowing its substrate to bind in the space between the loop and catalytic residues. Upon substrate binding (in Hyb-24DN-A(112)/Ald complex), the loop is shifted 4.3 A at Tyr170-C(alpha), and the side-chain of Tyr170 is rotated. By the combined effect, Tyr170-O(eta) moves a total of 10.5 A, resulting in the formation of hydrogen bonds with the nitrogen of amide linkage in Ald (closed form). In addition, electrostatic interaction between Asp181-O(delta) and the amino group in Ald stabilizes thesubstrate binding. We propose here that the enzyme catalysis proceeds according to the following steps: (i) Ald-induced transition from open to closed form, (ii) nucleophilic attack of Ser112 to Ald and formation of a tetrahedral intermediate, (iii) formation of acyl enzyme and transition to open form, (iv) deacylation. Amino acid substitutions reducing the enzyme/Ald interaction at positions 181 or 170 drastically decreased the Ald-hydrolytic activity, but had very little effect on esterolytic activity, suggesting that esterolytic reaction proceeds regardless of conversion. Present models illustrate why new activity against the nylon oligomer has evolved in an esterase with beta-lactamase folds, while retaining the original esterolytic functions.
机译：我们对6-氨基己酸二聚体水解酶（Hyb-24DN）进行了X射线晶体分析，Hyb-24DN是一种负责降解尼龙6的酶（一种工业副产品），也是Hyb-24DN-A之间的复合物（112） （Hyb-24DN的S112A突变体）和6-氨基己酸酯线性二聚体（Ald）的分辨率分别为1.58 A和1.4A。在Hyb-24DN中，Asp181-Oδ与Tyr170-Oeta形成氢键，其中两个催化和结合氨基酸位于Asn167和Val177之间。这种状态是所谓的开放形式，允许其底物结合在环和催化残基之间的空间中。底物结合后（在Hyb-24DN-A（112）/ Ald络合物中），环在Tyr170-Cα处移位4.3 A，并且Tyr170的侧链旋转。通过组合作用，Tyr170-O（eta）总共移动10.5 A，导致与Ald（封闭形式）的酰胺键氮形成氢键。另外，Asp181-Oδ与Ald中的氨基之间的静电相互作用稳定了底物的结合。我们在这里建议酶催化按照以下步骤进行：（i）Ald诱导的从开放形式到封闭形式的转变;（ii）Ser112对Ald的亲核攻击和四面体中间体的形成;（iii）酰基酶的形成并转变为开放形式，（iv）脱酰作用。氨基酸取代减少了181或170位的酶/ Ald相互作用，大大降低了Ald水解活性，但对酯水解活性影响很小，表明酯水解反应无论转化如何都进行。目前的模型说明了为什么针对尼龙低聚物的新活性在具有β-内酰胺酶折叠的酯酶中得以发展，同时又保留了原有的酯分解功能。

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