6-羟基烟酸3-单加氧酶(NicC)催化反应机理研究
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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,K_m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,K_m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。
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 Nic C,and then the His-tagged NicC was purified to homogeneity.The optimal temperature reaction range of Nic C 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 K_mand V_(max)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 microbia degradation of pyridinic compounds.
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 Nic C,and then the His-tagged NicC was purified to homogeneity.The optimal temperature reaction range of Nic C 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 K_mand V_(max)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 microbia degradation of pyridinic compounds.
引文
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[13]Zhang Y T,Chen Q,Ji J B,et al.Complete genome sequence of Alcaligenes faecalis strain JQ135,a bacterium capable of efficiently degrading nicotinic acid.Current Microbiology,2018,75(12):1551-1554.
[14]Nakano N,Fujisawa H.Purification,characterization and gene cloning of 6-hydroxynicotinate 3-monooxygenase from Pseudomonas fluorescens TN5.European Journal of Biochemistry,1999,260(1):120-126.
[15]Hurh B,Yamane T,Nagasawa T.Purification and characterization of nicotinic acid dehydrogenase from Pseudomonas fluorescens TN5.Journal of Fermentation and Bioengineering,1994,78(1):19-26.
[16]Hicks K A,Yuen M E,Feng Z W,et al.Structural and biochemical characterization of 6-hydroxynicotinic acid 3-monooxygenase,a novel decarboxylative hydroxylase involved in aerobic nicotinate degradation.Biochemistry,2016,55(24):3432-3446.
[17]Treiber N,Schulz G.Structure of 2,6-dihydroxypyridine 3-hydroxylase from a nicotine-degrading pathway.Journal of Molecular Biology,2008,379(1):94-104.
[18]Yu Hao,Robert P H,Tang H Z,et al.Mechanism of the 6-hydroxy-3-succinoyl-pyridine 3-monooxygenase flavoprotein from Pseudomonas putida S16.Journal of Biological Chemistry,2014,289(42):29158-29170.
[19]Gustafsson C,Govindarajan S,Minshull J.Codon bias and heterologous protein expression.Trends in Biotechnology,2004,22(7):346-353.
[2]Gerhild S,Lingens F.Bacterial degradation of N-heterocyclic compounds.Biochemistry of Microbial Degradation,1994,459-486.
[3]Kaiser J P,Feng Y C,Bollag J M.Microbial metabolism of pyridine,quinoline,acridine,and their derivatives under aerobic and anaerobic conditions.Microbiology Reviews,1996,60(3):483-498.
[4]Yu H,Tang H Z,Zhu X Y,et al.Molecular mechanism of nicotine degradation by a newly isolated strain,Ochrobactrum sp.Strain SJY1.Applied and Environmental Microbiology,2015,81(1):272-281.
[5]Yoshida T,Nagasawa T.Enzymatic functionalization of aromatic N-heterocycles:hydroxylation and carboxylation.Journal of Bioscience and Bioengineering,2000,89(2):111-118.
[6]Scriven E F V,Toomey J E,Murugan R.Pyridine and pyridine derivatives.Kirk-Othmer Encyclopedia of Chemical Technology,2005,20:1-33.
[7]Sims G K,O,Loughlin E J.Degradation of pyridines in the environment.Critical Reviews in Environmental Control,1989,19(4):309-340.
[8]Sims J,Lee S.Degradation of pyridine derivatives in soil.Journal of Environmental Quality,1985,14(4):580-584.
[9]胡春辉,徐青,于浩.Arthrobacter sp.2PR降解2-羟基吡啶动力学及降解特性研究.中国生物工程杂志,2017,37(8):31-38.Hu C H,Xu Q,Yu H.Characteristics and kinetic study of 2-hydroxypyridine degradation by a novel bacteria Arthrobacter sp.2PR.China Biotechnology,2017,37(8):31-38.
[10]Jimenez J I,Canales A,Jimenez-Barbero J,et al.Deciphering the genetic determinants for aerobic nicotinic acid degradation:the nic cluster from Pseudomonas putida KT2440.Proc Natl Acad Sci USA,2008,105(32):11329-11334.
[11]Tang H Z,Wang L J,Wang W W,et al.Systematic unraveling of the unsolved pathway of nicotine degradation in Pseudomonas.PLo S Genetics,2013,9(10):e1003923.
[12]Torimura M,Yoshida H,Kano K,et al.Bioelectrochemical transformation of nicotinic acid into 6-hydroxynicotinic acid on Pseudomonas fluorescens TN5-immobilized column electrolytic flow system.Journal of Molecular Catalysis B:Enzymatic,2000,8(4):265-273.
[13]Zhang Y T,Chen Q,Ji J B,et al.Complete genome sequence of Alcaligenes faecalis strain JQ135,a bacterium capable of efficiently degrading nicotinic acid.Current Microbiology,2018,75(12):1551-1554.
[14]Nakano N,Fujisawa H.Purification,characterization and gene cloning of 6-hydroxynicotinate 3-monooxygenase from Pseudomonas fluorescens TN5.European Journal of Biochemistry,1999,260(1):120-126.
[15]Hurh B,Yamane T,Nagasawa T.Purification and characterization of nicotinic acid dehydrogenase from Pseudomonas fluorescens TN5.Journal of Fermentation and Bioengineering,1994,78(1):19-26.
[16]Hicks K A,Yuen M E,Feng Z W,et al.Structural and biochemical characterization of 6-hydroxynicotinic acid 3-monooxygenase,a novel decarboxylative hydroxylase involved in aerobic nicotinate degradation.Biochemistry,2016,55(24):3432-3446.
[17]Treiber N,Schulz G.Structure of 2,6-dihydroxypyridine 3-hydroxylase from a nicotine-degrading pathway.Journal of Molecular Biology,2008,379(1):94-104.
[18]Yu Hao,Robert P H,Tang H Z,et al.Mechanism of the 6-hydroxy-3-succinoyl-pyridine 3-monooxygenase flavoprotein from Pseudomonas putida S16.Journal of Biological Chemistry,2014,289(42):29158-29170.
[19]Gustafsson C,Govindarajan S,Minshull J.Codon bias and heterologous protein expression.Trends in Biotechnology,2004,22(7):346-353.