Identification and Characterization of a Novel Gentisate 1,2-Dioxygenase Gene from a Halophilic Martelella Strain

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 (18)O 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.

Figure 1. Amino acid sequence analysis results.

(A) Neighbor-joining tree of the predicted GDO amino acid sequence. (B) Excerpts of GDO sequences used for multiple sequence alignment performed by Vector NTI program. Aligned sequences are from halophilic Martelella sp. AD-3 (WP_024706076), Corynebacterium glutamicum ATCC 13032 (NP_602217.1), Rhodococcus opacus R7 (ABH01038.1), Rhodococcus jostii RHA1 (ABG93677.1), Bradyrhizobium japonicum (NP_766750), Pseudomonas alcaligenes NCIB 9867 (xlnE) (AAD49427.1), Xanthobacter polyaromaticivorans (BAC98955.1), Klebsiella pneumoniae M5a1 (WP_004870415), Pseudomonas aeruginosa UCBPP-PA14 (ZP00135722), Sphingomonas sp. RW5 (CAA12267.1), Haloarcula sp. D1 (AAQ79814.1), Haloferax sp. D1227 (AAQ62856.1), Pseudomonas alcaligenes NCIB 9867 (hbzE) (ABD64513.1), Ralstonia (Pseudomonas) sp. U2 (AAD12619.1), Silicibacter (Ruegeria) pomeroyi DSS-3 (AAV97252.1). The positions of the four highly conserved His residues are indicated in the figures.

Figure 2. Full wavelength scanning results.

(A) SDS-PAGE analysis of expressed GDO in BL21 (DE3) on a 12.5% gel. Lane M, protein molecular weight marker (MBI); Lane 1, uninduced cells; Lane 2, cell culture induced with 1 mM IPTG; Lane 3, the supernatant of the induced cells; Lane 4, the precipitate of the induced cells; Lane 5, purified recombinant His₆-GDO protein; (B) NATIVE-PAGE of purified GDO-His₆. Lane M, protein molecular weight marker (MBI); Lane 1, 5 μl purified GDO-His₆.

Figure 3. Characterization of GDO.

(A) pH optimization of GDO. GDO has optimal activity at pH 7.0. (B) Temperature sensitivity of GDO enzyme activity; optimal activity was obtained at 30 °C. (C) Salinity-dependence of GDO activity; maximum enzyme activity was observed at 12%. (D) Effects of metal salts on enzyme activity; control, without metal salt. Ni, Ni²⁺; Co, Co²⁺; Ca, Ca²⁺; Cu, Cu²⁺; Mn, Mn²⁺; Zn, Zn²⁺; Mg, Mg²⁺; K, K⁺; Fe, Fe²⁺.

Figure 4. Enzyme kinetics of the purified GDO.

A mixture containing 0.46 mM gentisate in 0.1 M phosphate buffer, pH 7.4, was mixed with an equal volume of (2.4 mg/ml) GDO. Absorbance was measured at times ranging from 0 s to 10 s at 4 °C at the UV absorbance spectra, 330 nm.

Figure 5. ¹⁸O isotope experiments.

(A) Control experiment, unlabeled H₂O was used as a solvent and unlabeled O₂ was used for the reaction. (B) Labeled H₂¹⁸O was used as a solvent and unlabeled O₂ was used for reaction. (C) Unlabeled H₂O was used as a solvent and labeled ¹⁸O₂ was used for the reaction. (D) Labeled H₂¹⁸O was used as a solvent and labeled ¹⁸O₂ was used for the reaction.