基于量子力学分析β-内酰胺合成酶合成吡拉西坦中间体的机制

doi:10.11949/0438-1157.20240215

摘要/Abstract

摘要：

2-(氧代吡咯烷-1基)-乙酸(2-OAA)是合成吡拉西坦的重要前体，在医药合成领域起着关键作用。然而，目前尚未对2-OAA的生物合成方法和机制进行研究。为了填补这一研究空白，首先根据相似反应原则对β-内酰胺合成酶进行筛选，得到了一种来自于Streptomyces fulvorobeus的酶(SfAsnA)，该酶可以在水相中合成2-OAA，其酶活达到109.8 U/g，总转化数(TTN)可达79.02。随后，对SfAsnA进行蛋白模型构建与底物对接分析，以确定底物在酶活性中心的结合方式。然后，对合成2-OAA的本底反应机制进行详细分析，确定了目标反应的化学合成机制和势能面。最后，对酶促反应机制进行研究，并通过对催化双联体(Y254和E288)进行突变验证，进一步确定了SfAsnA催化2-OAA合成的机制以及该双联体在目标反应中的关键作用，为酶法合成2-OAA提供了理论基础。

关键词: 2-(氧代吡咯烷-1基)-乙酸, 生物催化, 内酰胺化, 分子模拟, 反应能垒, 计算化学

Abstract:

2-(2-Oxopyrrolidin-1-yl)-acetic acid (2-OAA) is an important precursor for the synthesis of piracetam and plays a significant role in the field of pharmaceutical synthesis. However, there is currently a lack of biological methods and reaction mechanism analysis for synthesizing it. To address this research gap, based on the principle of similarity reaction, the β-lactam synthetase (SfAsnA) from Streptomyces fulvorobeus was identified. This enzyme has the ability to synthesize 2-OAA in the aqueous phase, exhibiting an enzyme activity of 109.8 U/g and a total conversion number (TTN) of up to 79.02. Subsequently, the protein model construction of the SfAsnA was analyzed with the substrate to determine the combination of the substrate in the enzyme activity center. Next, the reaction mechanism of the non-enzymatic reaction for synthesizing 2-OAA was analyzed in detail to verify the chemical synthesis mechanism and relative energy profile of the target reaction. Finally, the enzymatic response mechanism is studied, and mutation verification is used to catalyze dual-combined bodies (Y254 and E288) to further determine the SfAsnA catalytic 2-OAA synthesis mechanism and the key role of the dual-connected body in the target response. It provides a solid theoretical foundation for enzyme synthesis of 2-OAA.

Key words: 2-(2-oxopyrrolidin-1-yl)-acetic acid, biocatalysis, lactamization, molecular simulation, reaction energy barrier, computational chemistry

中图分类号:

O 643.1

马文哲, 宋伟, 魏婉清, 吴静, 刘立明. 基于量子力学分析β-内酰胺合成酶合成吡拉西坦中间体的机制[J]. 化工学报, 2024, 75(12): 4712-4722.

Wenzhe MA, Wei SONG, Wanqing WEI, Jing WU, Liming LIU. Mechanism analysis of β-lactam synthase in synthesizing piracetam intermediate based on quantum mechanics[J]. CIESC Journal, 2024, 75(12): 4712-4722.

图/表 8

图1 叔酰胺及2-(氧代吡咯烷-1基)-乙酸的研究现状

Fig.1 Progress in the synthesis of tertiary amide and 2-(2-oxopyrrolidin-1-yl)-acetic acid

表1 SfAsnA突变体引物设计

Table 1 Primers design of SfAsnA mutant

Primer	Sequence (5′-3′)
Y254A-F	CGCATCCTCACCGGGGCGGGCGCG
Y254A-R	GGGGATGTCCGCGCCCGCCCCGGT
E288A-F	ACCTTCGACGGGCTGAACGCGATGTCC
E288A-R	GGACAGCACCGGGGACATCGCGTTCAG

图2 2-OAA的合成测试

Fig.2 Synthesis test of 2-OAA

图3 SfAsnA与INT1的对接结果和关键相互作用

Fig.3 The binding and key interactions between SfAsnA and INT1

图4 目标反应的机制预测与验证

Fig.4 Mechanism prediction and verification of target reaction

图5 SfAsnA催化目标反应的机制预测与验证

Fig.5 Mechanism prediction and verification of target reaction by SfAsnA

表2 SfAsnA及突变体内酰胺化4-CMBA的动力学参数

Table 2 Kinetic parameters of WT and its mutants for lactamization of 4-CMBA

酶	K_m/(mmol/L)	k_cat/min^-1	(k_cat/K_m)/(L/(mmol·min))
WT	9.5±1.27	(8.9±0.62)×10^-2	(9.4±1.41)×10^-3
Y254A	10.3±1.35	(1.0±0.07)×10^-2	(1.0±0.15)×10^-3
E288A	10.1±1.22	(1.2±0.08)×10^-2	(1.2±0.16)×10^-3

图6 SfAsnA催化目标反应的条件优化

Fig.6 Optimizing conditions of target reaction by SfAsnA

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