NDLI: Purification and characterization of salicylate 5-hydroxylase, a three-component monooxygenase from Ralstonia sp. strain U2

Content Provider	Springer Nature Link
Author	Fang, Ti Zhou, Ning Yi
Copyright Year	2013
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.
Starting Page	671
Ending Page	679
Page Count	9
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	98
Issue Number	2
e-ISSN	14320614
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2013-04-27
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Iron–sulfur protein Ralstonia sp. strain U2 Salicylate 5-hydroxylase Three-component oxygenase Microbiology Microbial Genetics and Genomics Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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