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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 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.
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Received: 23 November 2018
Published: 05 August 2019
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Corresponding Authors:
hao YU
E-mail: yuhaosunshine@163.com
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