Journal
FRONTIERS IN MICROBIOLOGY
Volume 9, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fmicb.2018.01335
Keywords
moderate halophilic bacteria; 3-hydroxybenzoate; 3-hydroxybenzoate 6-hydroxylase; purification; FAD binding
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Funding
- Research Projects of Environmental Protection Public Welfare Industry in China [201509035]
- National Natural Science Foundation of China [41301329, 51578240]
- Chinese National Science Foundation for Excellent Young Scholars [31422004]
- Yangtze River Delta Technology Projects of Shanghai Municipal Science and Technology Commission [17295810602]
- Excellent Team Program of Shanghai Academy of Agricultural Sciences [2017(A-03)]
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3-Hydroxybenzoate 6-hydroxylase, an NADH-dependent flavoprotein, can convert 3-hydroxybenzoate which is an important intermediate in the biodegradation of many aromatic hydrocarbons. 3-Hydroxybenzoate is metabolized by entering the TCA cycle through the gentisate pathway. We found a putative 3HB6H gene from a cluster that potentially encodes for gentisic acid degradation from a halophilic Martelella sp. strain AD-3. The corresponding protein was expressed with an N-terminal His-tag and purified by Ni2+-nitrilotriacetic acid affinity chromatography. The protein showed an overexpressed band of about 46 kDa by SDS-PAGE, and it was also proven that the enzyme contains FAD by absorption spectroscopy and HPLC analysis. The optimal activity of 3HB6H from strain AD-3 was observed in phosphate buffer (pH 8.0) at 37 degrees C without salinity (NaCl) and metal salts. The K-m values of 3-hydroxybenzoate 6-hydroxylase were determined to be 72.6 +/- 10.1 mu M and 104.1 +/- 18.2 mu M for 400 mu M NADH and 3-hydroxybenzoate, respectively. Site-directed mutagenesis showed that residues 305, 306 and 308 are important for FAD binding. In addition, we found that Tyr221 and Gln305 of 3HB6H from strain AD-3 are involved in substrate binding.
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