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 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.

Mimicking the Aromatic‐Ring‐Cleavage Activity of Gentisate‐1,2‐Dioxygenase by a Nonheme Iron Complex

“…[2] Gentisic acid (2,5-dihydroxybenzoic acid) is ak ey intermediate in the biodegradation of mono-and poly-cyclic aromatic,a nd hetero-aromatic compounds by microorganisms. [3,4] Gentisate-1,2-dioxygenase (GDO), [5][6][7][8][9][10] the nonheme iron enzyme isolated from avariety of sources,c atalyzes the aromatic C À Cb ond cleavage of gentisate to form maleyl pyruvate using dioxygen as the oxidant (Scheme 1). Ther ing cleavage product maleyl pyruvate is further degraded by direct hydrolysis to maleate and pyruvate or undergoes isomerization to form fumaryl pyruvate which hydrolyzes to form fumarate and pyruvate, the central metabolites of the Krebs cycle.…”

“…Although there are several reports on the characterization and biochemical studies of this enzyme, [3,5,10,13,17] functional model of GDO exhibiting the oxidative CÀCb ond cleavage of gentisate has not been developed yet. Moreover,t he mechanistic details of this enzymatic reaction remain unexplored.…”

Mimicking the Aromatic‐Ring‐Cleavage Activity of Gentisate‐1,2‐Dioxygenase by a Nonheme Iron Complex

“…Gentisic acid (2,5‐dihydroxybenzoic acid) is a key intermediate in the biodegradation of mono‐ and poly‐cyclic aromatic, and hetero‐aromatic compounds by microorganisms . Gentisate‐1,2‐dioxygenase (GDO), the nonheme iron enzyme isolated from a variety of sources, catalyzes the aromatic C−C bond cleavage of gentisate to form maleyl pyruvate using dioxygen as the oxidant (Scheme ). The ring cleavage product maleyl pyruvate is further degraded by direct hydrolysis to maleate and pyruvate or undergoes isomerization to form fumaryl pyruvate which hydrolyzes to form fumarate and pyruvate, the central metabolites of the Krebs cycle .…”

“…Although there are several reports on the characterization and biochemical studies of this enzyme, functional model of GDO exhibiting the oxidative C−C bond cleavage of gentisate has not been developed yet. Moreover, the mechanistic details of this enzymatic reaction remain unexplored.…”

A Structural Model for the Iron–Nitrosyl Adduct of Gentisate Dioxygenase

“…Although the structure of GDO from some bacteria have been confirmed,, the understanding of its catalytic cycle still remains unclear . Recently, GDO from halophilic bacteria, Martelella strain AD‐3, isolated from highly saline petroleum‐contaminated soil, has been reported …”