Fig 1.
(A) Metabolic networks of nopaline, octopine, nopalinic acid, and octopinic acid. NOS and OCS also synthesize nopalinic acid and octopinic acid, respectively [5, 11]. The inhibition experiment performed in this study revealed that nopalinic acid and octopinic acid may be active substrates for flavin-containing dehydrogenases for nopaline and octopine, respectively (dashed arrow). (B) Schematic gene clusters related to the metabolism of opines by bacteria. Homologous genes are indicated in the same color and correspond to Fig 1A. Gray genes are putative opine transporters.
Fig 2.
Characterization of opine dehydrogenase from P. putida.
(A) Plasmid construction for the expression of PpOdhABC genes in P. putida cells. The inset shows the nucleotide sequences of the PpOdhB, PpOdhC, and PpOdhA 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 PpOdhA 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 E. coli 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.
Table 1.
Kinetic parameters for PpOpnDH.
Fig 3.
Inhibition study by α-keto acids (A) and L-amino acids (B) of PpOpnDH.
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 (http://rsb.info.nih.gov/ij/).
Fig 4.
Characterization of opine dehydrogenases from B. japonicum.
(A) A Western blotting analysis. BjOdhB2+A+C, BjOdhB1+A+C, and BjOdhB2+B1+A+C proteins were overexpressed in E. coli 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 Odh 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 Fig 2F; upper), and extracts of BjOdhAB2C (middle) and BjOdhAB1C (lower). Numbers with peaks are the molar ratio of FAD:FMN.
Table 2.
Kinetic parameters for BjOpnDH2.
Table 3.
Kinetic parameters for BjOpnDH1.
Fig 5.
Phylogenetic analysis using β- (A) and α-subunits (B) of flavin-containing OpnDH.
Letters in parentheses are the GenBankTM accession numbers. The number on each branch indicates the bootstrap value. Proteins with asterisks were used for Fig 6.
Fig 6.
Partial multiple sequence alignment of deduced amino acid sequences of β- (A), γ- (B), and α-subunits (C) of flavin-containing OpnDH.
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-X-Gly-X2-Gly) and [2Fe-2S]-binding motifs (Cys-X4-Cys-X2-Cys-X11~12-Cys and Cys-X-Cys-X15~20-Cys-X4-Cys) are shaded in red, orange, yellow, and cyan, respectively. FMN-binding sites are shaded in green.