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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (7): 15-23    DOI: 10.13523/j.cb.20190703
研究报告     
6-羟基烟酸3-单加氧酶(NicC)催化反应机理研究 *
王菲,胡春辉,于浩()
青岛农业大学生命科学学院 山东省应用真菌重点实验室 青岛 266109
Catalytic Mechanism of 6-Hydroxynicotinic Acid 3-Monooxygenase (NicC)
Fei WANG,Chun-hui HU,hao YU()
Shandong Provincial Key Laboratory of Applied Mycology, College of Life Science, Qingdao Agricultural University, Qingdao 266109, China
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摘要：

恶臭假单胞菌(Pseudomonas putida)KT2440中的6-羟基烟酸(6HNA)3-单加氧酶(NicC)是烟酸代谢过程中的关键酶。NicC通过在吡啶环上加羟基对吡啶环进行活化,从而使吡啶环可在双加氧酶催化下开环,最终被完全降解。通过去除NicC的N端稀有密码子增加了NicC的表达量,进一步利用Ni-Sepharose重力柱对NicC进行了纯化。通过实验发现,NicC的最适反应温度为30~40℃,最适反应pH为8.0。Cd 2+对NicC的酶活有明显的抑制作用。当NADH的浓度为0.25mmol/L时,底物6HNA所对应的NicC的最大酶活为14.1U/mg,Km值为51.8μmol/L;当6HNA的浓度为0.25mmol/L时,底物NADH所对应的NicC的最大酶活为10.79U/mg,Km值为15.0μmol/L。通过HPLC和LC-MS分析表明,NicC可以在NADH和氧气的参与下催化6HNA转化生成2,5-二羟基吡啶(2,5-DHP)和甲酸,还可以将对羟基苯甲酸转化生成对苯二酚。同位素标记实验表明,产物2,5-DHP中的氧原子来源于参与反应的氧气。为研究吡啶类化合物微生物代谢提供了理论基础。

关键词： 6-羟基烟酸3-羟化酶;  酶学性质;  催化机制;  同位素标记    
Abstract:

6-Hydroxynicotinic acid (6HNA) 3-monooxygenase (NicC) is the key enzyme for nicotinic degradation in Pseudomonas putida KT2440. NicC can catalyze the hydroxylation of pyridine ring to promote the ring cleavage reaction of pyridine ring. The expression level of NicC was enhanced by replace the rare codon in the N-terminal of NicC, and then the His-tagged NicC was purified to homogeneity. The optimal temperature reaction range of NicC is from 30℃ to 40℃, and the optimal reaction pH is 8.0. The Cd 2+ could significantly inhibit the activity of NicC. The apparent Km and Vmax values of the purified NicC for 6HNA were 51.8μmol/L and 14.1U/mg, respectively, and those for NADH were 15.0μmol/L and 10.79U/mg, respectively. According to the HPLC and LC-MS analysis, NicC could catalyzes 6HNA to form 2,5-DHP and formic acid, and it could also transform 4-hydroxybenzoic acid to form hydroquinone. Isotope labeling experiments proved that the oxygen atom incorporated into 2,5-DHP is from dioxygen. The study will provide useful information for the microbial degradation of pyridinic compounds.

Key words: 6-Hydroxynicotinic acid 3-monooxygenase    Enzymatic properties    Catalytic mechanism    Isotope labeling experiments
收稿日期: 2018-11-23 出版日期: 2019-08-05
ZTFLH:  Q819  
基金资助: * 国家自然科学基金青年项目(31600086);山东省自然科学基金青年基金资助项目(ZR2016CQ06)
通讯作者: 于浩     E-mail: yuhaosunshine@163.com
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引用本文:

王菲,胡春辉,于浩. 6-羟基烟酸3-单加氧酶(NicC)催化反应机理研究 *[J]. 中国生物工程杂志, 2019, 39(7): 15-23.

Fei WANG,Chun-hui HU,hao YU. Catalytic Mechanism of 6-Hydroxynicotinic Acid 3-Monooxygenase (NicC). China Biotechnology, 2019, 39(7): 15-23.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190703    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I7/15

图1  NicC进化树分析
图2  NicC蛋白的表达纯化
图3  NicC催化反应的HPLC分析
图4  NADH还原后的还原态NicC的全波长扫描图谱
图5  NicC催化反应的光谱学分析
图6  NicC酶学性质研究
图7  NicC催化PHB转化的HPLC分析
图8  NicC催化反应的同位素标记实验
图9  NicC催化反应的同位素标记实验
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