Characterization of gentisate 1,2-dioxygenase HagA from Haloferax volcanii WFD11

Abstract:

[Objective] We studied the growth characteristics of Haloferax volcanii WFD11 cultivated on a number of aromatic acids as carbon sources to reveal the possible difference between bacteria and archaea in aromatic acids degradation. [Methods] The putative gentisate 1,2-dioxygenase gene was cloned and the enzyme heterologously expressed, purified and functionally identified before determining its key enzyme parameters. We measured the growth of H. volcanii WFD11 in halophile growth medium minimal medium containing one of the six aromatic acids (4 mmol/L) separately as sole carbon source. The intermediate from 3-hydroxybenzoic acid catabolism was determined using high performance liquid chromatography. The putative gentisate 1,2-dioxygenase gene, designated hagA, was heterologously expressed in H. volcanii H1424, and HagA was purified by affinity chromatography on an Ni2+ chelating column of rapid purification system. HagA was functionally identified based on its specific activity to catalyze the ring cleavage of gentisate using ultraviolet spectrophotometer. The expression type of hagA was revealed by real-time PCR. [Results] Strain WFD11 could use 4 mmol/L 3-hydroxybenzoic acid (3HBA) and 3-hydroxyphenylpropionic acid (3HPP) as sole carbon source for growth, but not on 4 mmol/L benzoic acid (BA), 2-hydroxybenzoic acid (2HBA), 4-hydroxybenzoic acid (4HBA), 3-phenylpropionic (3PP). Gentisate was determined as the intermediate during 3HBA catabolism. The cell extract of H. volcanii WFD11 catalyzed the ring cleavage of gentisate to maleylpyruvate. Purified HagA exhibited a specific activity of 0.024 8 U/mg of gentisate 1,2-dioxygenase, without Fe2+. Real-time PCR proved that hagA was constitutively expressed in strain WFD11. [Conclusion] This study provides the basis to further explore the difference between bacteria and archaea in aromatic acids degradation.