Chiral NAD analogs as cofactors for biocatalysis

doi:10.16085/j.issn.1000-6613.2021-0553

Abstract

Abstract:

Nicotinamide adenine dinucleotide (NAD) is an essential redox cofactor in life. Albeit there are two anomers, namely β- and α-NAD, of which the anomeric carbon atom linked to the nicotinamide moiety holds an R- and S-configuration, respectively, most NAD-dependent enzymes strongly prefer β-NAD. Recently, simplified NAD analogs have been synthesized and explored as redox cofactors for biocatalysis. The fact that many synthetic NAD analogs are achiral promotes us to wonder whether enzymes discriminate between NAD analog enantiomers. Herein, two pairs of NAD analog enantiomers were prepared through Zincke reaction by using readily available amino-3-phenylpropanates as chiral sources, and tested as cofactors for mutants of cytochrome P450 monooxygenase from Bacillus megaterium (P450 BM3 R966D/W1046S) and glucose dehydrogenase from Sulfolobus solfataricus (SsGDH I192T/V306I). Results showed that P450 BM3 R966D/W1046S had much higher catalytic efficiencies when coupled with the reduced forms of the S-configuration analogs. This preliminary study suggested that amino acids may be used as chiral sources to prepare NAD analogs with defined stereochemistry and that more chiral NAD analogs should be explored to match redox enzymes and their engineered variants.

Key words: biocatalysis, cofactor, nicotinamide adenine dinucleotide (NAD) analog, stereochemistry, redox enzyme

摘要：

烟酰胺腺嘌呤二核苷酸（NAD）是生命体中必不可少的氧化还原辅因子。NAD具有β-NAD和α-NAD两种差向异构体，而大多数NAD依赖酶偏好β-NAD。近年来，已合成大量结构简化的NAD类似物，并探索其作为氧化还原辅因子应用于生物催化反应。通常认为酶天然具有立体选择性，而很多结构简化的NAD类似物不含手性中心，暗示有必要合成手性NAD类似物以评估其性能。本研究拟设计合成手性NAD类似物，并初步探讨部分氧化还原酶利用它们作为辅酶的催化性能。以商品化光学纯的氨基-3-苯基丙酸为手性原料，合成了两对NAD类似物对映体，并用来源于巨芽孢杆菌的突变型细胞色素P450单加氧酶（P450 BM3 R966D/W1046S）和来源于硫化叶菌的突变型葡萄糖脱氢酶（SsGDH I192T/V306I），测试这些NAD类似物作为辅酶的催化活性。结果发现，P450 BM3 R966D/W1046S利用还原型S构型NAD类似物的催化效率高于对应的R构型NAD类似物；而SsGDH I192T/V306I则对NAD类似物对映体均没有活性。研究初步表明，氨基酸可作为手性原料制备立体构型明确的NAD类似物，而氧化还原酶表现出偏好特定手性NAD类似物的现象，也提示需要合成更多的手性NAD类似物，并系统测试和匹配氧化还原酶及其突变体，以创建出基于辅酶依赖型氧化还原酶的新催化体系。

关键词: 生物催化, 辅酶, 烟酰胺腺嘌呤二核苷酸类似物, 立体化学, 氧化还原酶

CLC Number:

Q552

LI Qing, LIU Wujun, GUO Xiaojia, WANG Qian, ZHAO Zongbao. Chiral NAD analogs as cofactors for biocatalysis[J]. Chemical Industry and Engineering Progress, 2021, 40(9): 5214-5221.

李青, 刘武军, 郭潇佳, 王倩, 赵宗保. 手性NAD类似物合成及其辅酶应用[J]. 化工进展, 2021, 40(9): 5214-5221.

Figures/Tables 6

References 39

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辅因子	K_m/mmol?L^-1	K_cat/s^-1	(K_cat?K_m)/mmol^-1?L?s^-1
L-PheNAH^①	0.40±0.06	0.07±0.00	0.17±0.02
D-PheNDH^①	1.06±0.13	0.06±0.00	0.05±0.00
3S-PheNAH^①	0.41±0.11	0.02±0.00	0.04±0.01
3R-PheNAH^①	0.22±0.07	0.00±0.00	0.01±0.00
BNAH^①	0.23±0.04	0.10±0.01	0.42±0.05
β-NADH^①	0.27±0.06	0.21±0.01	0.78±0.12
BNAH^②	0.07±0.01	0.09±0.00	1.25±0.11
β-NADH^②	0.16±0.03	0.20±0.01	1.31±0.18

辅因子	K_m/mmol?L^-1	K_cat/s^-1	(K_cat?K_m)/mmol^-1?L?s^-1
L-PheNAH^①	0.40±0.06	0.07±0.00	0.17±0.02
D-PheNDH^①	1.06±0.13	0.06±0.00	0.05±0.00
3S-PheNAH^①	0.41±0.11	0.02±0.00	0.04±0.01
3R-PheNAH^①	0.22±0.07	0.00±0.00	0.01±0.00
BNAH^①	0.23±0.04	0.10±0.01	0.42±0.05
β-NADH^①	0.27±0.06	0.21±0.01	0.78±0.12
BNAH^②	0.07±0.01	0.09±0.00	1.25±0.11
β-NADH^②	0.16±0.03	0.20±0.01	1.31±0.18

辅因子	酶活/U?mg^-1	辅因子	酶活/U?mg^-1
L-PheNA	—	D-PheNA	—
3S-PheNA	—	3R-PheNA	—
BNA	1.7×10^-3	β-NAD	0.84

辅因子	酶活/U?mg^-1	辅因子	酶活/U?mg^-1
L-PheNA	—	D-PheNA	—
3S-PheNA	—	3R-PheNA	—
BNA	1.7×10^-3	β-NAD	0.84

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