Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria

Abstract

Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium 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 noxB-noxA and ooxB-ooxA 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 Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A), 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 Bradyrhizobium japonicum consisted of OdhB 1 -C-A-B 2, 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.

Characterization of iron-sulfur clusters in flavin-containing opine dehydrogenase

“…The specific activity and k cat /K m values of the αβγ C61S mutant were 0.257 unit/mg protein and 118 min −1 ·mM −1 , respectively, which were half of those of αβγ WT (0.504 unit/mg protein and 224 min −1 ·mM −1 ). 2) On the other hand, the α C382S βγ and αβ mutants both had~10-fold lower specific activities: 0.0285 and 0.0363 unit/mg protein, respectively. These results suggest that the modification of [Fe-S] site 2 had significant effects not only on structural folding, but also enzyme catalysis, and that the γ-subunit by itself (but not [Fe-S] site 1 ) was necessary for enzyme activity.…”

“…Although difficulties have been associated with expressing the dehydrogenase(s) from A. tumefaciens, 1) we recently functionally expressed homologous proteins from the non-pathogenic bacteria, Pseudomonas putida KT2440 and Bradyrhizobium japonicum USDA110. 2) The novel opine dehydrogenase (OpnDH) belongs to a group of so-called "dye-linked dehydrogenases" that catalyze the oxidation of various organic acids, amino acids, and alcohols in the presence of an artificial electron acceptor, such as 2,6-dichloroindophenol (Cl2Ind), in which FAD and/or FMN is commonly contained as a prosthetic group(s). This enzyme is phylogenetically related to hydrogen cyanide synthase (EC 1.4.99.5; Hcn) from bacteria, 3) D-hydroxyproline dehydrogenase (HypDH) from bacteria, 4,5) and L-proline dehydrogenase (EC 1.5.5.2; ProDH) from archaea, [6][7][8][9] with a heterooligomeric structure, the β-subunit of which functions as a catalytic subunit by itself ( Fig.…”

“…The bll7191, bll7190, and bll7192 genes, encoding the α-, β-, and γ-subunits of BjOpnDH, respectively, were introduced into pETDuet-1, pACYCDuet-1, and pCOLADuet-1 (Novagen), by which the (His) 6 -tag was attached to the N-terminus of the bll7190 gene, whereas the S-tag was attached to the C-termini of the bll7191 and bll7192 genes. 2) In order to destroy [Fe-S] site 1 or [Fe-S] site 2 , each cysteine residue codon (TGC) at the positions of 61 and 382 in the γand α-subunits, respectively, was replaced with serine (TCC) using the single-round PCR method. Wild-type (αβγ WT ) and mutated BjOpnDH proteins were expressed in Escherichia coli BL21(DE3) and purified as described previously.…”

“…Wild-type (αβγ WT ) and mutated BjOpnDH proteins were expressed in Escherichia coli BL21(DE3) and purified as described previously. 2) OpnDH activity was measured by monitoring the reduction of Cl2Ind at 600 nm at 30°C in a reaction mixture with the following composition: 50 mM Tris-HCl buffer (pH 9.0), 0.05 mM Cl2Ind, and 1 mM octopine. One unit was defined as the amount of the enzyme that catalyzed the reduction of 1 μmol of Cl2Ind/min.…”

“…Prosthetic groups from~3 mg/ml BjOpnDH protein were extracted as described previously. 2) Numbers with peaks are the molar ratio of FAD:FMN, compared with the standards (data not shown). (D).…”