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

Analgesic Effect of Passive Range-of-Motion Exercise on the Healthy Side for Pain after Total Knee Arthroplasty: A Prospective Randomized Trial

Background. Exercise can reduce the pain threshold momentarily and induce analgesia, which is called exercise-induced hypoalgesia (EIH). Exercise therapy for inducing EIH may be an effective treatment option for pain. We aimed at investigating whether continuous passive motion (CPM) on both healthy and affected sides could induce EIH and reduce pain in the operated knee in patients after unilateral total knee arthroplasty (TKA). Patients and Methods. In this prospective randomized controlled trial, participants were randomly assigned to two groups: a bilateral group that received bilateral exercise on the operated and healthy sides and a unilateral group that received exercise therapy only on the affected side. We enrolled 40 patients aged ≥60 years who were scheduled to undergo unilateral TKA. Visual analogue scale (VAS) scores and range of motion (ROM) on the operated side were measured immediately before and after CPM on postoperative days 2, 4, 7, and 14. The primary outcome was the difference in the VAS scores before and after CPM on postoperative day 14. The secondary outcome was the difference in the ROM before and after CPM on postoperative day 14. Results. Comparison of VAS scores before and after CPM showed no significant intergroup differences on all measurement dates. However, there was a significant difference in values on day 14 (P<0.05). Both groups showed an increase in ROM after CPM, with significant increments observed on days 2 and 4 in the bilateral group and on day 14 in the unilateral group. There was no significant difference in values on postoperative day 14. Conclusion. Post-TKA pain was reduced by performing the same exercise on the healthy knee during CPM therapy. This could be due to EIH, and the results indicated that EIH can also influence postoperative pain immediately after surgery

Characterization of <i>cis</i>-4-hydroxy-D-proline dehydrogenase from <i>Sinorhizobium meliloti</i>

<p>The <i>hypO</i> gene from <i>Sinorhizobium meliloti</i>, located within the <i>trans</i>-4-hydroxy-L-proline metabolic gene cluster, was first successfully expressed in the host <i>Pseudomonas putida</i>. Purified HypO protein functioned as a FAD-containing <i>cis</i>-4-hydroxy-D-proline dehydrogenase with a homomeric structure. In contrast to other known enzymes, significant activity for D-proline was found, confirming a previously proposed potential involvement in D-proline metabolism.</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