Abstract
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 18O isotope experiment and LC-MS analysis, the sources of the two oxygen atoms added onto maleylpyruvate were identified as H2O and O2, respectively. The Km and kcat values for gentisic acid were determined to be 26.64 μM 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.
Highlights
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
Amino acid sequence of the AD-3 shared 30.9% and 29.1% sequence identities with two amino acid sequences from Pseudomonas alcaligenes NCIB 98677
A phylogenetic tree was constructed with GDO proteins from 15 other strains and demonstrated that the protein from strain AD-3 is most closely related to Rhodococcus opacus R73 and Rhodococcus jostii RHA115 (Fig. 1B)
Summary
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. 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. Comparative studies of the characterization of different gentisate dioxygenases from moderately halophilic bacteria will contribute to the comprehension of the limitations of degrading of PAHs. Martelella sp. Strain AD-3, a moderate halophilic bacterium, was isolated from highly saline petroleum-contaminated soil in Shandong province, China[12] It is highly effective in degrading many PAHs, such as naphthalene, anthracene, and phenanthrene, under broad salinities (0.1–15%) and varying pH (6.0–10.0)[13]. The source of the two oxygen atoms added to the maleylpyruvate was analyzed
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