Catalytic Mechanism of 6-Hydroxynicotinic Acid 3-Monooxygenase (NicC)

doi:10.13523/j.cb.20190703

China Biotechnology

2019, Vol. 39

Issue (7): 15-23 DOI: 10.13523/j.cb.20190703

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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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 K_m and 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 microbial degradation of pyridinic compounds.

Key words： 6-Hydroxynicotinic acid 3-monooxygenase Enzymatic properties Catalytic mechanism Isotope labeling experiments

Received: 23 November 2018 Published: 05 August 2019

ZTFLH:

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Corresponding Authors: hao YU E-mail: yuhaosunshine@163.com

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Cite this article:

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

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

Fig.1 Phylogenetic analysis of NicC and related hydroxylases The accession number of each protein was indicated in parenthesis. The bar represents 0.2 amino acid substitutions per site

Fig.2 Expression and purification of NicC (a) The rare codon (AGG) in the N-terminal of nicC gene was changed to CGC (b) Spectrum of NicC and NicC + 1% SDS (c) SDS-PAGE image of purified His-tagged NicC M: Protein marker; NicC-O: Unmodified NicC; NicC-M: Modified NicC protein

Fig.3 HPLC analysis of NicC catalyzed reaction (a) NicC was mixed with 1mmol/L NADH and 1mmol/L 6HNA for 5min (b) NicC was mixed with and 1mmol/L 6HNA for 5min

Fig.4 Spectrum of NicC reduced by excess NADH

Fig.5 Spectrophotometric analysis of NicC catalyzed reaction Left curve (Line1) showed the NicC catalyzed reaction with 0.25mmol/L 6HNA and NADH; Line 2: Reaction mixture of 0.125mmol/L formic acid+0.125mmol/L NAD⁺+formate dehydrogenase; Line3:Formate dehydrogenase was added into NicC catalyzed reaction at 300s; Line4: Formate dehydrogenase+0.125mmol/L NAD⁺; Line5:NicC catalyzed reaction without adding any substrates

Fig.6 Characterization of NicC (a) Temperature dependent enzyme activity of NicC (b) Temperature stability of NicC (c) pH-dependent enzyme activity of NicC (d) Effects of metal ions on NicC activity (e) Kinetic studies of NicC for 6HNA (f) Kinetic studies of NicC for NADH

Fig.7 HPLC analysis of NicC catalyzed reaction with PHB (a) HPLC analysis of NicC catalyzed reaction with 1mmol/L NADH and 1mmol/L PHB (b) The HPLC signal of 1mmol/L hydroquinone

Fig.8 Isotope labeling experiments of NicC catalyzed reaction (a) MS analysis of NicC catalyzed reaction in

H 2 16

O (b) MS analysis of NicC catalyzed reaction in

H 2 18

Fig.9 Isotope labeling experiments of NicC catalyzed reaction (a) MS analysis of NicC catalyzed reaction in ¹⁶O₂ (b) MS analysis of NicC catalyzed reaction in ¹⁸O₂


[1]	Xu P, Yu B, Li F L , et al. Microbial degradation of sulfur, nitrogen and oxygen heterocycles. Trends in Microbiology, 2006,14(9):398-405. doi: 10.1016/j.tim.2006.07.002

[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. doi: 10.1128/AEM.02265-14

[5]	Yoshida T, Nagasawa T . Enzymatic functionalization of aromatic N-heterocycles: hydroxylation and carboxylation. Journal of Bioscience and Bioengineering, 2000,89(2):111-118. doi: 10.1016/S1389-1723(00)88723-X

[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. doi: 10.1080/10643388909388372

[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.

[9]	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. doi: 10.1073/pnas.0802273105

[11]	Tang H Z, Wang L J, Wang W W , et al. Systematic unraveling of the unsolved pathway of nicotine degradation in Pseudomonas. PLoS Genetics, 2013,9(10):e1003923. doi: 10.1371/journal.pgen.1003923

[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. doi: 10.1016/S1381-1177(99)00077-6

[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. doi: 10.1007/s00284-018-1486-0

[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. doi: 10.1046/j.1432-1327.1999.00124.x

[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. doi: 10.1016/0922-338X(94)90172-4

[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. doi: 10.1021/acs.biochem.6b00105

[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. doi: 10.1016/j.jmb.2008.03.032

[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. doi: 10.1074/jbc.M114.558049

[19]	Gustafsson C, Govindarajan S, Minshull J . Codon bias and heterologous protein expression. Trends in Biotechnology, 2004,22(7):346-353. doi: 10.1016/j.tibtech.2004.04.006

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