Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria

<div><p>Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with <i>Agrobacterium</i> species, and are synthesized by well-studied NAD(P)H-dependent dehydrogenases (synthases), which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti) plasmid. In addition to the reverse oxidative reaction(s), the genes <i>noxB-noxA</i> and <i>ooxB</i>-<i>ooxA</i> are considered to be involved in opine catabolism as (membrane-associated) oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation). We herein successfully expressed Nox/Oox-like genes from <i>Pseudomonas putida</i> in <i>P</i>. <i>putida</i> cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the <i>OdhA</i>, <i>OdhB</i>, and <i>OdhC</i> genes, which were arranged in tandem on the chromosome (<i>OdhB-C-A</i>), and exhibited dehydrogenase (but not oxidase) activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from <i>Bradyrhizobium japonicum</i> consisted of <i>OdhB</i>_<i>1</i><i>-C-A-B</i>_<i>2</i>, from which two proteins, OdhAB₁C and OdhAB₂C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB₁ and OdhB₂ in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by “subunit-exchange”. To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase.</p></div

Partial multiple sequence alignment of deduced amino acid sequences of β- (A), γ- (B), and α-subunits (C) of flavin-containing OpnDH.

<p>White letters in black boxes indicate highly conserved amino acid residues. N-terminal ~120 amino acid residues of the α-subunit of L-ProDH corresponding to the γ-subunit of flavin-containing OpnDH and D-HypDH. Two ADP-binding motifs (Gly-<i>X</i>-Gly-<i>X</i>₂-Gly) and [2Fe-2S]-binding motifs (Cys-<i>X</i>₄-Cys-<i>X</i>₂-Cys-<i>X</i>_11~12-Cys and Cys-<i>X</i>-Cys-<i>X</i>_15~20-Cys-<i>X</i>₄-Cys) are shaded in red, orange, yellow, and cyan, respectively. FMN-binding sites are shaded in green.</p

Kinetic parameters for BjOpnDH₁.

<p>^a Under standard assay conditions described in the Materials and Methods section.</p><p>^b Not determined due to trace activity.</p><p>Kinetic parameters for BjOpnDH₁.</p

Characterization of opine dehydrogenase from <i>P</i>. <i>putida</i>.

<p>(A) Plasmid construction for the expression of <i>PpOdhABC</i> genes in <i>P</i>. <i>putida</i> cells. The inset shows the nucleotide sequences of the <i>PpOdhB</i>, <i>PpOdhC</i>, and <i>PpOdhA</i> genes at the intergenic regions along with the corresponding deduced amino acid sequences. N-terminal amino acid sequences, determined using the purified enzyme (Fig 2B), are indicated by underlining. SI, X, Ba, Bg, H, and E indicate SalI, XhoI, BamHI, BglI, HindIII, and EcoRI restriction enzyme sites, respectively. The BglI site within the <i>PpOdhA</i> gene was removed without changing the amino acid sequence. (B) An SDS-PAGE analysis of purified recombinant PpOdhABC (20 μg in 15% (w/v) gel). (C) Absorption spectra using 10 mg/ml enzyme solution (inset). (D) A zymogram staining analysis used nopaline (lanes 1 and 2) and octopine (lane 3) as substrates together with the PMS/INT (lanes 1 and 3) and PMS/NBT assay systems (lane 2). Purified enzymes of 50 μg were applied. (E) Functional characterization of α-, β-, and γ-subunits, corresponding to PpOdhA, PpOdhB, and PpOdhC, respectively. All (His)₆-tagged proteins were expressed in <i>E</i>. <i>coli</i> cells, and purified using a Ni-NTA column. Prosthetic groups were analyzed by HPLC (Fig 2F). (F) Elution profiles of the standard mixture of FAD and FMN (upper) and extract of PpOdhABC (lower). Numbers with peaks are the molar ratio of FAD:FMN.</p

Kinetic parameters for PpOpnDH.

<p>^a Under standard assay conditions described in the Materials and Methods section.</p><p>Kinetic parameters for PpOpnDH.</p

Characterization of opine dehydrogenases from <i>B</i>. <i>japonicum</i>.

<p>(A) A Western blotting analysis. BjOdhB₂+A+C, BjOdhB₁+A+C, and BjOdhB₂+B₁+A+C proteins were overexpressed in <i>E</i>. <i>coli</i> cells, and purified by a Ni-NTA column using the (His)₆-tag attached to BjOdhB₂ (for BjOdhB₂+A+C and BjOdhB₂+B₁+A+C) or BjOdhB₁ (for BjOdhB₁+A+C), whereas S-tag was attached to other <i>Odh</i> genes. After SDS-PAGE of 20 μg protein per lane (Fig 4B), antibodies against the N-terminal (His)₆-tag (left panel) and C-terminal S-tag (light panel) were used for immunoblotting. (B) An SDS-PAGE analysis of purified recombinant BjOdhAB₂C and BjOdhAB₁C. (C) Absorption spectra using 10 mg/ml BjOdhAB₂C (red) and BjOdhAB₁C (blue) solution (inset). (D) A HPLC analysis of prosthetic groups. Elution profiles of the standard mixture (the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138434#pone.0138434.g002" target="_blank">Fig 2F</a>; upper), and extracts of BjOdhAB₂C (middle) and BjOdhAB₁C (lower). Numbers with peaks are the molar ratio of FAD:FMN.</p

Phylogenetic analysis using β- (A) and α-subunits (B) of flavin-containing OpnDH.

<p>Letters in parentheses are the GenBank^TM accession numbers. The number on each branch indicates the bootstrap value. Proteins with asterisks were used for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138434#pone.0138434.g006" target="_blank">Fig 6</a>.</p

Kinetic parameters for BjOpnDH₂.

<p>^a Under standard assay conditions described in the Materials and Methods section.</p><p>^b Not determined due to trace activity.</p><p>Kinetic parameters for BjOpnDH₂.</p

Inhibition study by α-keto acids (A) and L-amino acids (B) of PpOpnDH.

<p>Nopaline (0.1 mM) was used as a substrate. Relative specific activity values were expressed as percentages of the values obtained in the absence of an inhibitor. IC₅₀ values were calculated by curve fitting using ImageJ software (<a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>).</p