Purification and crystallization of 6-hydroxynicotinic acid 3-monooxygenase NicC

Abstract:

[Background] N-heterocyclic aromatic compounds (NHACs) are one of the important environmental pollutants. Long-term accumulation in the human body can lead to diseases. The degradation of N-heterocyclic aromatic compounds by microorganism is an effective approach. [Objective] The 6-hydroxynicotinic acid 3-monooxygenase gene nicC was cloned and expressed in Escherichia coli, the NicC protein was purified and the crystallization conditions were studied. [Methods] The nicC gene was amplified from the genomic DNA of Pseudomonas putida KT2440 and a recombinant expression vector pET28a-nicC was constructed and expressed in E. coli BL21(DE3). Affinity and gel filtration chromatography were used to purify NicC. The preliminary screening and optimization of crystals were done by using hanging drop diffusion method. [Results] The pET28a-nicC was constructed successfully and the purified NicC protein was obtained. Through preliminary screening of crystallization conditions and orthogonal optimization experiments, the optimal crystallization conditions were 0.2 mol/L NH4Cl, 0.01 mol/L CaCl2·2H2O, 31% PEG4000, 0.05 mol/L Tris-HCl ph 8.0, 4 °C for NicC, and 0.2 mol/L NH4Cl, 0.01 mol/L CaCl2·2H2O, 0.05 mol/L Tris-HCl ph 7.9, 31% PEG3350, 4 °C for SeMet-NicC and cocrystals of NicC and 6-HNA. [Conclusion] The construction of NicC protein purification system and study of crystallization conditions provided favorable conditions for the final analysis of the three-dimensional structure of NicC protein. The results laid a foundation for revealing the molecular mechanism of the pyridine ring to β-position monooxygenase to recognize the pyridine ring-containing substrate and catalyze the hydroxylation of β-position on the pyridine ring of the substrate.