腈水解酶立体选择性改造及其合成布瓦西坦

doi:10.11949/0438-1157.20240356

摘要/Abstract

摘要：

布瓦西坦是新一代抗癫痫药物，具有广阔的市场前景。利用腈水解酶立体选择性催化3-氰基己腈合成关键手性中间体（R）-3-氰基己酸，进一步经加氢、环化、手性拆分等合成布瓦西坦路线，具有原子经济性高等优势，然而目前挖掘的腈水解酶存在立体选择性差等缺陷。以腈水解酶PgNIT为研究对象，通过对其催化口袋和亚基界面改造获得了立体选择性大幅提高的突变体F164W/I202R，产物光学纯度由野生型的86%提升至97%，E值由17提高至111。分子动力学模拟研究表明，F164位点的改造减小了（R）-3-氰基己腈深入催化口袋内部的空间位阻，而“A”界面I202位点的改造增加了亚基之间缔合的距离和催化口袋区域的原子动态协同性，从而显著提升了腈水解酶的立体选择性。

关键词: 腈水解酶, 立体选择性, 分子改造, 分子模拟, 生物催化, 分子生物学

Abstract:

Brivaracetam is a new generation of anti-epileptic drugs with broad market prospects. The nitrilase-catalyzed synthesis of (R)-3-cyanohexanoic acid from 3-cyanocapronitrile, followed by hydrogenation, cyclization and chiral resolution to synthesize brivaracetam is a promising route due to the high atomic economy. However, the poor enantioselectivity of the exploited nitrilases has strongly restricted its application. In this study, the nitrilase PgNIT from Paraburkholderia graminis was exploited. The catalytic pocket and subunit interface domain were modified and a mutant F164W/I202R with significantly improved enantioselectivity was obtained. As a result, the ee_p value of F164W/I202R was increased from 86% to 97%, while the E value rose from 17 to 111 compared to wild type, respectively. Molecular dynamics simulation analyses revealed that the mutation of F164 reduced the steric hindrance on the entry of (R)-3-cyanocapronitrile into the catalytic pocket. On the other hand, the modification of I202 site at the “A” interface increased the association distance between subunits and increased the dynamic coordination of atoms in the catalytic pocket domain, thus significantly improved the enantioselectivity.

Key words: nitrilase, enantioselectivity, molecular modification, molecular simulation, biocatalysis, molecular biology

中图分类号:

Q 814.4

吴哲明, 张碧云, 郑仁朝. 腈水解酶立体选择性改造及其合成布瓦西坦[J]. 化工学报, 2024, 75(7): 2633-2643.

Zheming WU, Biyun ZHANG, Renchao ZHENG. Engineering of nitrilase enantioselectivity for efficient synthesis of brivaracetam[J]. CIESC Journal, 2024, 75(7): 2633-2643.

图/表 16

图1 化学-腈水解酶法合成布瓦西坦路线

Fig.1 Chemical-enzymatic route for synthesis of brivaracetam from 3-cyanohexanitrile

表1 上下游引物

Table 1 Primers used in this work

Primer	Sequence（5′ to 3′）
T133-F	ACCCCANNKCACTTCGAACGTATGATTT
T133-R	ACGTTCGAAGTGMNNTGGGGTGATAAA
E136-F	ACCCCAACTCACTTCNNKCGTATGATTTGGG
E136-R	CCCCAAATCATACGMNNGAAGTGAGTTGGG
F164-F	CTGGCGTGTNNKGAACATAACAACCCA
F164-R	TTATGTTCGAAMNNCGCCAGCTGACCA
P188-F	TTCTGCGATGTATNNKGGCAGCGCTTTTG
P188-R	CAAAAGCGCTGCCMNNATACATCGCAGA
F196-F	TGGCGAAGGTNNKGCGCAGCGTATGGAA
F196-R	TACGCTGCGCMNNACCTTCGCCAAAAGC
E201-F	GCGCAGCGTATGNNKATCAACATTCGTC
E201-R	GACGAATGTTGATMNNCATACGCTGCGC
I202-F	GCAGCGTATGGAANNKAACATTCGTCAGCC
I202-R	GGCCTGACGAATGTTMNNTTCCATACGCTG
N203-F	CGTATGGAAATCNNKATTCGTCAGCACG
N203-R	CGTGCTGACGAATMNNGATTTCCATACG
I204-F	ATGGAAATCAACNNKCGTCAGCACGCAC
I204-R	GTGCGTGCTGACGMNNGTTGATTTCCAT
R205-F	GAAATCAACATTNNKCAGCACGCACTGG
R205-R	CCAGTGCGTGCTGMNNAATGTTGATTTC
Q206-F	AAATCAACATTCGTNNKCACGCACTGGA
Q206-R	TCCAGTGCGTGMNNACGAATGTTGATTT

图2 分子动力学模拟PgNIT催化口袋示意图

Fig.2 Catalytic pocket of stable conformation obtained from molecular dynamics simulation

图3 PgNIT四聚体的界面示意图

Fig.3 Interface diagram of PgNIT tetramer

图4 PgNIT四聚体上“A”界面相交的关键Loop环

Fig.4 Key Loop on “A” interface in PgNIT tetramer

图5 F164位点的饱和突变结果

Fig.5 Relative activity and enantioselectivity of mutants at F164 site

图6 F164W/I202位点的饱和突变结果

Fig.6 Relative activity and enantioselectivity of mutants at F164W/I202 site

图7 腈水解酶PgNIT及其突变体的SDS-PAGE分析

Fig.7 SDS-PAGE analysis of PgNIT and its mutant

表2 PgNIT及其突变体的动力学参数

Table 2 Kinetic parameters of PgNIT and variants towards 3-cyanocapronitrile

突变体	K_m/(mmol/L)	K_cat/min^-1	(K_cat/K_m)/ (L/(mmol·min))
PgNIT	1.38±0.19	21.37±1.80	15.48
F164W	0.44±0.06	12.91±0.79	29.34
F164W/I202R	1.68±0.42	6.99±1.22	4.16

表3 PgNIT及其突变体的立体选择性、催化活性及热稳定性

Table 3 Kinetic parameters of PgNIT and variants towards 3-cyanocapronitrile

突变体	E	比酶活/（U/mg）	半衰期t_1/2/h
PgNIT	18±4	2.48±0.06	63.3±0.61
F164W	30±2	3.47±0.11	55.5±1.71
F164W/I202R	112±5	1.29±0.06	39.7±2.22

图8 腈水解酶PgNIT及其突变体在30℃的半衰期

Fig.8 Half lives of purified PgNIT and its mutants at 30℃

图9 PgNIT和F164W/I202R催化(R)-3-氰基己腈和(S)-3-氰基己腈的底物结合腔示意图

Fig.9 Illustration of substrate-binding cavities of PgNIT and its mutant F164W/I202R

图10 PgNIT和F164W/I202R突变体的蛋白模型叠合图（黄色部分为C163，表示催化口袋位置；粉红色和浅蓝色Loop环为F164W/I202R的“A”界面关键Loop环；紫色和深蓝色Loop环为PgNIT的关键Loop环）

Fig.10 Illustration of protein model unity of PgNIT and its mutant F164W/I202R

图11 F164W和F164W/I202R突变体的DCCM结果

Fig.11 Dynamics cross-correlation map for C α atom pairs within F164W and F164W/I202R

图12 PgNIT及其突变体的亲核进攻距离示意图

Fig.12 Comparison and analysis of differences between enzyme-substrate binding conformations for PgNIT and mutants

图13 PgNIT和F164W/I202R催化3-氰基己腈的反应进程

Fig.13 Time course of 3-cyanocapronitrile hydrolysis by PgNIT and F164W/I202R at different substrate concentration

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