TY - JOUR
T1 - Identification and characterization of a novel gentisate 1,2-dioxygenase gene from a halophilic martelella strain
AU - Huang, Ling
AU - Hu, Haiyang
AU - Tang, Hongzhi
AU - Liu, Yongdi
AU - Xu, Ping
AU - Shi, Jie
AU - Lin, Kuangfei
AU - Luo, Qishi
AU - Cui, Changzheng
PY - 2015/9/23
Y1 - 2015/9/23
N2 - Halophilic Martelella strain AD-3, isolated from highly saline petroleum-contaminated soil, can efficiently degrade polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene and anthracene, in 3-5% salinity. Gentisic acid is a key intermediate in the microbial degradation of PAH compounds. However, there is little information on PAH degradation by moderately halophilic bacteria. In this study, a 1,077-bp long gene encoding gentisate 1,2-dioxygenase (GDO) from a halophilic Martelella strain AD-3 was cloned, sequenced, and expressed in Escherichia coli. The recombinant enzyme GDO was purified and characterized in detail. By using the 18 O isotope experiment and LC-MS analysis, the sources of the two oxygen atoms added onto maleylpyruvate were identified as H 2 O and O 2, respectively. The K m and k cat values for gentisic acid were determined to be 26.64 €‰ 1/4M and 161.29 €‰s '1, respectively. In addition, optimal GDO activity was observed at 30 €‰°C, pH 7.0, and at 12% salinity. Site-directed mutagenesis demonstrated the importance of four highly conserved His residues at positions 155, 157, 167, and 169 for enzyme activity. This finding provides new insights into mechanism and variety of gentisate 1,2-dioxygenase for PAH degradation in high saline conditions.
AB - Halophilic Martelella strain AD-3, isolated from highly saline petroleum-contaminated soil, can efficiently degrade polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene and anthracene, in 3-5% salinity. Gentisic acid is a key intermediate in the microbial degradation of PAH compounds. However, there is little information on PAH degradation by moderately halophilic bacteria. In this study, a 1,077-bp long gene encoding gentisate 1,2-dioxygenase (GDO) from a halophilic Martelella strain AD-3 was cloned, sequenced, and expressed in Escherichia coli. The recombinant enzyme GDO was purified and characterized in detail. By using the 18 O isotope experiment and LC-MS analysis, the sources of the two oxygen atoms added onto maleylpyruvate were identified as H 2 O and O 2, respectively. The K m and k cat values for gentisic acid were determined to be 26.64 €‰ 1/4M and 161.29 €‰s '1, respectively. In addition, optimal GDO activity was observed at 30 €‰°C, pH 7.0, and at 12% salinity. Site-directed mutagenesis demonstrated the importance of four highly conserved His residues at positions 155, 157, 167, and 169 for enzyme activity. This finding provides new insights into mechanism and variety of gentisate 1,2-dioxygenase for PAH degradation in high saline conditions.
UR - https://www.scopus.com/pages/publications/84942235855
U2 - 10.1038/srep14307
DO - 10.1038/srep14307
M3 - 文章
C2 - 26394696
AN - SCOPUS:84942235855
SN - 2045-2322
VL - 5
JO - Scientific Reports
JF - Scientific Reports
M1 - 14307
ER -