title: Purification and characterization of salicylate 5-hydroxylase, a three-component monooxygenase from Ralstonia sp. strain U2.
abstract: Salicylate is an important intermediate in the bacterial degradation of polycyclic aromatic hydrocarbons and salicylate hydroxylases play essential roles in linking the peripheral and ring-cleavage catabolic pathways. Unlike the well-characterized salicylate 1-hydroxylases, the rarely occurred salicylate 5-hydroxylase (S5H) has not been characterized in detail. In this study, the three-component Fe-S protein complex (NagAaGHAb) of S5H from Ralstonia sp. strain U2 was purified, and its biochemical and catalytic properties were characterized. The oxygenase component NagGH exhibited an α₃β₃ heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per α subunit. NagAa is the ferredoxin-NADP⁺ reductase component containing flavin and plant type [2Fe-2S] cluster. The ferredoxin component NagAb was characterized as a [2Fe-2S] dimer which remains remarkably stable in denaturing gel electrophoresis after being heated at 100 °C for 1 h. Purified NagAa and NagAb, NagGH catalyzed the hydroxylation of salicylate to gentisate with a specific activity of 107.12 ± 14.38 U/g and showed an apparent K(m) for salicylate of 102.79 ± 27.20 μM and a similar K(m) value for both NADH and NADPH (59.76 ± 7.81 μM versus 56.41 ± 12.76 μM). The hydroxylase exhibited different affinities for two hydroxysalicylates (2,4-dihydroxybenzoate K(m) of 93.54 ± 18.50 μM versus 2,6-dihydroxybenzoate K(m) of 939.80 ± 199.46 μM). Interestingly, this S5H also showed catalytic activity to the pollutant 2-nitrophenol and exhibited steady-state kinetic data of the same order of magnitude as those for salicylate. This study will allow further comparative studies of structure-function relationships of the ring hydroxylating mono- and di-oxygenase systems.
authors.affiliation: Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China.
authors.forename: Ti
authors.initials: T
authors.lastname: Fang
authors.forename: Ning-Yi
authors.initials: NY
authors.lastname: Zhou
mesh_terms: D005841:Gentisates / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D055503:Protein Multimerization; D043367:Ralstonia / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D005841:Gentisates; D006899:Mixed Function Oxygenases; C099614:salicylic acid 5-hydroxylase