Dataset Information

Purification and Initial Characterization of 3-Hydroxybenzoate 6-Hydroxylase From a Halophilic Martelella Strain AD-3.

ABSTRACT: 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°C without salinity (NaCl) and metal salts. The K_m values of 3-hydroxybenzoate 6-hydroxylase were determined to be 72.6 ± 10.1 ?M and 104.1 ± 18.2 ?M for 400 ?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.

SUBMITTER: Chen X

PROVIDER: S-EPMC6043778 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Purification and Initial Characterization of 3-Hydroxybenzoate 6-Hydroxylase From a Halophilic Martelella Strain AD-3.

Chen Xin X Tang Hongzhi H Liu Yongdi Y Xu Ping P Xue Yong Y Lin Kuangfei K Cui Changzheng C

Frontiers in microbiology 20180706

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 ...[more]

PMID: 30034371

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Dataset Information

Purification and Initial Characterization of 3-Hydroxybenzoate 6-Hydroxylase From a Halophilic Martelella Strain AD-3.

Publications

Purification and Initial Characterization of 3-Hydroxybenzoate 6-Hydroxylase From a Halophilic <i>Martelella</i> Strain AD-3.

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