Inverse Virtual Screening, Recombinant Expression, and Activity Characterization of Purine Degrading Enzymes

LIU Yu; ZHANG Xinxin; WANG Rui; MENG Xianghong

doi:10.13386/j.issn1002-0306.2024030464

Volume 46 Issue 5

Feb. 2025

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Science and Technology of Food Industry > 2025 > 46(5): 145-152. > DOI: 10.13386/j.issn1002-0306.2024030464

LIU Yu, ZHANG Xinxin, WANG Rui, et al. Inverse Virtual Screening, Recombinant Expression, and Activity Characterization of Purine Degrading Enzymes[J]. Science and Technology of Food Industry, 2025, 46(5): 145−152. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2024030464.

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Inverse Virtual Screening, Recombinant Expression, and Activity Characterization of Purine Degrading Enzymes

College of Food Science and Engineering, Ocean University of China, Qingdao 266404, China

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Received Date: March 31, 2024
Available Online: December 28, 2024

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Abstract

Abstract

To develop a novel purine degrading enzyme that can be used in the food industry, this study aimed to screen an enzyme with potential purine degradation activity using inverse virtual screening technology and molecular docking technology, clone express, purify, and validate the activity of this enzyme, and explore the optimal reaction conditions for its degradation of purines. The results showed that the docking binding energies of dihydroorotase screened by the inverse virtual screening platform PharmMapper with adenine, guanine, hypoxanthine, and xanthine were −7.3, −6.9, −7.6, and −8.8 kcal/mol, respectively, which had the optimum docking result. The dihydroorotase with a molecular weight of 41 kDa was obtained with cloning expression. The optimal conditions for catalyzing the degradation of guanine were pH8.0, 25 ℃, and under the catalysis of Zn²⁺ (0.5 mmol/L), guanine could be degraded at a ratio of 46.1%. Therefore, it found that dihydroorotase had the degradation activity for guanine, and the optimal reaction conditions for guanine degradation were determined, which laid foundation for the application in degradation of food purine.
- inverse virtual screening,
- molecular docking,
- cloning expression,
- guanine,
- degradation

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References (33)

References

[1]	BORTOLOTTI M, POLITO L, BATTELLI M G, et al. Xanthine oxidoreductase:One enzyme for multiple physiological tasks[J]. Redox Biology,2021,41:101882. doi: 10.1016/j.redox.2021.101882
[2]	郑敏. 猪肉嘌呤含量表型变异及其遗传基础研究[D]. 南昌:江西农业大学, 2017. [ZHENG M. Phenotypic variation and genetic basis of purine content in pork[D]. Nanchang:Jiangxi Agricultural University, 2017.] ZHENG M. Phenotypic variation and genetic basis of purine content in pork[D]. Nanchang: Jiangxi Agricultural University, 2017.
[3]	于婷婷, 程翅, 喻田. 尿酸肠道代谢的研究现状[J]. 中国临床药理学杂志,2021,37(21):2978−2984. [YU T T, CHENG C, YU T. Research status of uric acid intestinal metabolism[J]. The Chinese of Clinical Pharmacology,2021,37(21):2978−2984.] YU T T, CHENG C, YU T. Research status of uric acid intestinal metabolism[J]. The Chinese of Clinical Pharmacology, 2021, 37（21）: 2978−2984.
[4]	DU F J, HUANG Y Q, HAN R F, et al. Prevalence and trends of hyperuricemia among adults over 40 years old in an economically developed region of China from 2017 to 2020[J]. Journal of Public Health,2022,32(1):43−51.
[5]	中国民族卫生协会重症代谢疾病分会. 中国高尿酸血症相关疾病诊疗多学科专家共识(2023年版)[J]. 中国实用内科杂志,2023,6(43):461−480. [Chinese National Health Association of Severe Metabolic Diseases Branch. Chinese multidisciplinary expert consensus on the diagnosis and treatment of hyperuricemia-related diseases (2023)[J]. Chinese Journal of Practical Internal Medicine,2023,6(43):461−480.] Chinese National Health Association of Severe Metabolic Diseases Branch. Chinese multidisciplinary expert consensus on the diagnosis and treatment of hyperuricemia-related diseases （2023）[J]. Chinese Journal of Practical Internal Medicine, 2023, 6（43）: 461−480.
[6]	LU J, SUN W Y, CUI L L, et al. Across-sectional study on uric acid levels among Chinese adolescents[J]. Pediatric Nephrology,2020,35:441−446. doi: 10.1007/s00467-019-04357-w
[7]	SHA W Q, HOU C L, YUAN E, et al. Different processed milk with residual xanthine oxidase activity and risk of increasing serum uric acid level[J]. Food Bioscience,2021,40:100892. doi: 10.1016/j.fbio.2021.100892
[8]	CHOI H K, ATKINSON K, KARLSON E W, et al. Purine-rich foods, dairy and protein intake, and the risk of gout in men[J]. The New England Journal of Medicine,2004,350(11):1093−1103. doi: 10.1056/NEJMoa035700
[9]	WANG F J, SUN L, ZONG G, et al. Associations of amino acid and acylcarnitine profiles with incident hyperuricemia in middle-aged and older Chinese individuals[J]. Arthritis Care & Rearch,2020,72(9):1305−1314.
[10]	CHEN D D, LI Q X, WU J C. Efficient removal of purine compounds from solutions via biomass carbons derived from pomelo peel[J]. Journal of Bioscience and Bioengineering,2023,136(5):383−390. doi: 10.1016/j.jbiosc.2023.09.002
[11]	WANG X L, YANG S J, LU J, et al. Screening and application of purine degrading Limosilactobacillus fermentum LF-1 from Huangjiu fermentation broth[J]. Journal of the Science of Food and Agriculture,2023,103(15):7921−7931. doi: 10.1002/jsfa.12881
[12]	谢微. 适合痛风病人食用的低嘌呤脱脂豆腐粉的研制[D]. 大庆:黑龙江八一农垦大学, 2012. [XIE W. Development of low purine defatted tofu powder suitable for gout patients[D]. Daqing:Heilongjiang Bayi Agricultural University, 2012.] XIE W. Development of low purine defatted tofu powder suitable for gout patients[D]. Daqing: Heilongjiang Bayi Agricultural University, 2012.
[13]	李慧慧. 低嘌呤豆浆及其速溶粉的研究[D]. 贵阳:贵州大学, 2016. [LI H H. Study on low purine soybean milk and its instant powder[D]. Guiyang:Guizhou University, 2016.] LI H H. Study on low purine soybean milk and its instant powder[D]. Guiyang: Guizhou University, 2016.
[14]	潘婷婷, 韦智, 李佳钰, 等. 浸泡时间对豆浆品质的影响及相关性分析[J]. 食品工业科技,2024,45(9):45−53. [PAN T T, WEI Z, LI J Y, et al. The effect of soaking time on the quality of soybean milk and its correlation analysis[J]. Technology of Food Industry,2024,45(9):45−53.] PAN T T, WEI Z, LI J Y, et al. The effect of soaking time on the quality of soybean milk and its correlation analysis[J]. Technology of Food Industry, 2024, 45（9）: 45−53.
[15]	JANKOWSKA D A, FAULWASSER K, TRAUTWEIN-SCHULT A, et al. Arxula adeninivorans recombinant adenine deaminase and its application in the production of food with low purine content[J]. Journal of Applied Microbiology,2013,115(5):1134−1146. doi: 10.1111/jam.12317
[16]	TRAUTWEIN-SCHULT A, JANKOWSKA D, CORDES A, et al. Arxula adeninivorans recombinant guanine deaminase and its application in the production of food with low purine content[J]. Journal of Molecular Microbiology and Biotechnology,2014,24(2):67−81.
[17]	JANKOWSKA D A, TRAUTWEIN-SCHULT A, CORDES A, et al. Arxula adeninivorans xanthine oxidoreductase and its application in the production of food with low purine content[J]. Journal of Applied Microbiology,2013,115(3):796−807. doi: 10.1111/jam.12284
[18]	LIU X F, OUYANG S S, YU B, et al. PharmMapper Server:A web server for potential drug target identification via pharmacophore mapping approach[J]. Nucleic Acids Research,2010,38:W609−W614. doi: 10.1093/nar/gkq300
[19]	WANG X, PAN C X, GONG J Y, et al. Enhancing the enrichment of pharmacophore-based target prediction for the polypharmacological profiles of drugs[J]. Journal of Chemical Information and Modeling,2016,56(6):1175−1183. doi: 10.1021/acs.jcim.5b00690
[20]	WANG X, SHEN Y H, WANG S W, et al. PharmMapper 2017 update:A web server for potential drug target identification with a comprehensive target pharmacophore database[J]. Nucleic Acids Research,2017,45(W1):W356−W360. doi: 10.1093/nar/gkx374
[21]	EBERHARDT J, SANTOS-MARTINS D, TILLACK A F, et al. AutoDock Vina 1.2. 0:New docking methods, expanded force field, and python bindings[J]. Journal of Chemical Information and Modeling,2021,61(8):3891−3898. doi: 10.1021/acs.jcim.1c00203
[22]	TROTT O, OLSON A J. AutoDock Vina:Improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading[J]. Journal of Computational Chemistry,2010,31(2):455−461. doi: 10.1002/jcc.21334
[23]	曲欣. 水产品中嘌呤含量分布及其在贮藏加工中变化规律的研究[D]. 青岛:中国海洋大学, 2013. [QU X. Study on the distribution of purine content in aquatic products and its variation during storage and processing[D]. Qingdao:Ocean University of China, 2013.] QU X. Study on the distribution of purine content in aquatic products and its variation during storage and processing[D]. Qingdao: Ocean University of China, 2013.
[24]	SHAMSOL AZMAN A N S, TAN J J, ABDULLAH M N H, et al. Network pharmacology and molecular docking analysis of active compounds in Tualang honey against atherosclerosis[J]. Foods,2023,12(9):1779. doi: 10.3390/foods12091779
[25]	王烨. A型H1N1流感病毒神经氨酸酶天然产物抑制剂的计算机模拟研究[D]. 长春:吉林大学, 2012. [WANG Y. Computer simulation of natural product inhibitors of neuraminidase of influenza A H1N1 virus[D]. Changchun:Jilin University, 2012.] WANG Y. Computer simulation of natural product inhibitors of neuraminidase of influenza A H1N1 virus[D]. Changchun: Jilin University, 2012.
[26]	PORTER T N, LI Y C, RAUSHEL F M. Mechanism of the dihydroorotase reaction[J]. Biochemistry,2004,43:16285−16292. doi: 10.1021/bi048308g
[27]	LI N, CUI R, ZHANG F, et al. A novel enzyme from Rhodotorula mucilaginosa aldolase:Isolation, identification and degradation for patulin in apple juice[J]. Process Biochemistry,2022,116:148−156. doi: 10.1016/j.procbio.2022.03.001
[28]	ZHOU L, LIU W, TEREFE N S. The inactivation kinetics of soluble and membrane-bound polyphenol oxidase in pear during thermal and high-pressure processing[J]. Food and Bioprocess Technology,2018,11(5):1039−1049. doi: 10.1007/s11947-018-2070-0
[29]	TIWARI K, KUMAR R, DUBEY V K. Biochemical characterization of dihydroorotase of Leishmania donovani:Understanding pyrimidine metabolism through its inhibition[J]. Biochimie,2016,131:45−53. doi: 10.1016/j.biochi.2016.09.009
[30]	TRUONG L, HEVENER K E, RICE A J, et al. High-level expression, purification, and characterization of Staphylococcus aureus dihydroorotase (PyrC) as a cleavable His-SUMO fusion[J]. Protein Expression and Purification,2013,88(1):98−106. doi: 10.1016/j.pep.2012.11.018
[31]	陆钰. 褐藻胶裂解酶的高效表达及酶学性质分析[D]. 无锡:江南大学, 2022. [LU Y. High-efficiency expression and enzymatic properties analysis of alginate lyase[D]. Wuxi:Jiangnan University, 2022.] LU Y. High-efficiency expression and enzymatic properties analysis of alginate lyase[D]. Wuxi: Jiangnan University, 2022.
[32]	王成华, 邢新会. 黄嘌呤氧化酶的研究进展及其发展前景[J]. 广西科学,2017,24(1):15−24. [WANG C H, XING X H. Research progress and development prospect of xanthine oxidase[J]. Guangxi Sciences,2017,24(1):15−24.] WANG C H, XING X H. Research progress and development prospect of xanthine oxidase[J]. Guangxi Sciences, 2017, 24（1）: 15−24.
[33]	SHI P, ZHANG R, LIU C X, et al. Computer-assisted in vitro reconstitution of purine degradation pathway to lower the purine content in food[J]. Journal of the Science of Food and Agriculture,2022,102(15):6787−7413. doi: 10.1002/jsfa.11289

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