The so-called dye-linked dehydrogenases catalyze the oxidation of various biomolecules in the presence of an artificial electron acceptor, in which several unique compounds related to plants as substrates such as opine(s) and L-hydroxyproline are contained. Opines including nopaline and octopine are produced from nutrients of plant by pathogenic agrobacteria species in a crown gall tumor and subsequently degraded for their nutrients by (hypothetical) opine dehydrogenase(s) (OpnDH). The homologous proteins of Pseudomonas putida and Bradyrhizobium japonicum (isozymes 1 and 2) function as nopaline- and octopine-specific dye-linked dehydrogenases, to yield α-ketoglutarate + L-arginine and pyruvate + L-arginine, respectively. L-Hydroxyproline is detected in such hydroxyproline-rich glycoprotein of plant cell walls. In the degradation pathway of bacteria, D-hydroxyproline dehydrogenase (HypDH) catalyzes the dehydrogenation reaction of cis-4-hydroxy-D-proline, and is classified into two types: homomeric and heteromeric enzymes. Both OpnDH and heteromeric HypDH commonly consist of three different subunits (αβγ), in which 2 FAD, 1 FMN, [2Fe-2S] and [4Fe-4S] clusters are contained as prosthetic groups. In D-amino acid dehydrogenase superfamily, these enzymes are physiologically related to L-proline dehydrogenase from archaea and hydrogen cyanide synthase from bacteria, whereas isozyme 2 of OpnDH from B. japonicum and other OpnDHs had appeared by convergent evolution.
- dye-linked dehydrogenase
- molecular evolution
Oxidoreductases catalyze the reversible electron-transfer from many compounds such as amino acids, alcohols, sugars, and amines, and are classified into three main groups, based on available electron acceptor(s): oxidases, oxygenases and dehydrogenases/reductases. FAD- and/or FMN-dependent dehydrogenases, one of subgroups in dehydrogenases/reductases, play a variety of important roles in the generation of energy and in the biosynthesis and metabolism of biomolecules [1, 2]. These enzymes are frequently called as “dye-linked dehydrogenases”, because of utilization of artificial dyes such as 2,6-dichloroindophenol (Cl2Ind) and ferricyanide instead of the natural acceptor(s). Furthermore, most of them are frequently associated with cell or organelle membranes and are unstable in solution, which has made their expression in host cells, purification, preservation, and characterization very difficult. Since dye-linked dehydrogenation activity for several unique compounds related to plants as substrates including opine(s) and
2. Opine dehydrogenase
2.1. Opine concept
Plant tumors known as crown gall are incited by pathogenic, soil-inhabiting Agrobacterium species including Agrobacterium tumefaciens. During the infections , the expression of the virulence genes (vir) within the tumor-inducing (Ti) plasmid is first specifically induced by phenolic compounds, including acetosyringone, which are released from the wounded plant cells (① in Figure 1). Next, the products expressed by the virD locus promote fragmentation of so-called T-DNA region (②) and subsequently transfer to the nuclei of host plants (③). T-DNA region integrated into a host chromosome contains several genes including synthase(s) of phytohormones such as auxin and cytokinin (④), and the genetic expressions direct the production of phytohormones such as auxin and cytokinin, which can lead to uncontrolled cell proliferation, producing a crown gall tumor (⑤). The (integrated) T-DNA region also encodes genes for the synthesis of unique compounds, opines (⑥). The opines produced and secreted by the neoplastic plant cells are utilizable as nutrient sources by the agrobacteria that induced the tumor (⑦). The genes for the utilization of the opines by the bacteria are also encoded by the Ti plasmid, but they are located outside the T-DNA region (⑧) (i.e., the “opine concept”).
2.2. What’s opine?
Opines have been structurally classified into several groups, among which two groups have a common secondary amine dicarboxylic acid structure. One group has been categorized as the N2-(l-
2.3. Opine assimilation by Agrobacterium
The octopine catabolic (occ) operons of octopine-type Ti plasmids consist of at least 15 genes that include ABC-type (opine) permease (encoded by occQMPJ) and ornithine cyclodeaminase (ocd)  (Figure 2). On the other hand, nopaline-metabolizing genes including nos are encoded within the nopaline catabolic (noc) region in the pTiC58 plasmid . Both the occ operon and noc region also contain several genes suspected of being involved in opine metabolism such as ooxA (pTi_037), ooxB (pTi_038), noxA (Atu6019), and noxB (Atu6021) (Figure 2A). In both opines, the first step of degradation is the reverse of biosynthesis, i.e., oxidative cleavage to
2.4. Nopaline dehydrogenase from Pseudomonas putida
2.4.1. Successful preparation of recombinant protein
The homologous ooxB-ooxA and noxB-noxA genes exist as a gene cluster together with putative ABC-type transporter gene(s) on the genomes and/or plasmids of non-Agrobacterium bacterial species, although the mechanisms underlying opine catabolism are unclear. Among them, PP_4457 and PP_4456 (genes) from Pseudomonas putida KT2440 correspond to OoxB (40% of identity) and NoxB (40%), and OoxA (41%) and NoxA (48%), respectively (referred to as OdhB and OdhA, respectively) (Figure 2A). The odhB-odhA operon, in which the (His)6-tag sequence was attached at the N-terminal of the odhB gene, expressed in P. putida cell (but not E. coli) [13, 14], and purified using immobilized metal (Ni2+) affinity chromatography (Figure 3A). A buffer system containing Tween-20 was absolutely necessary for this procedure, indicating tight binding to the cytoplasmic membrane. The purified recombinant protein consisted of three major distinct bands with molecular masses of 46, 42, and 9 kDa, respectively, among which the two formers expectedly corresponded to OdhA and OdhB, respectively. Interestingly, N-terminal sequence of the latter was identical with that of a provisional open-reading frame between odhA and odhB genes (referred to as odhC gene): the 3′ part of odhB and odhC was slightly overlapped by the 5′ part of odhC and odhA, respectively. Thus, this protein indeed consisted of α-, β-, and γ-subunits encoded by odhA, odhB, and odhC genes, respectively (referred to as PpOdhABC).
2.4.2. Prosthetic group(s)
The purified protein was orange-brown, and the absorption spectrum showed the characteristics of a typical flavoprotein (maxima at approximately 350 and 450 nm; Figure 3A): the flavin compounds were identified as FAD and FMN by HPLC. Alternatively, each subunit of heteromeric PpOdhABC was also purified and functionally characterized. Only FAD was extracted from (almost) inactive OdhA, OdhB, OdhAB (co-expression of OdhA+B), OdhBC, and OdhAC, while FAD and FMN were only extracted from active OdhABC; the molar ratio of FAD:FMN was 1.9:1.0. Therefore, it was concluded that PpOdhABC contained 2 FAD (α- and β-subunits) and 1 FMN (between the α- and β-subunits) within the structural unit of αβγ (Figure 4A). On the other hand, as described below in detail, the OdhC and OdhA proteins contain two different types of [Fe-S]-binding motifs, in which [4Fe-4S] and [2Fe-2S] clusters might be bound, respectively (see Section 2.6).
2.4.3. Characterization as a dye-linked opine dehydrogenase
When Cl2Ind was used as an artificial electron acceptor, the catalytic efficiency (kcat/Km) value of PpOdhABC with nopaline was 11,000-fold higher than that with octopine. p-Iodonitrotetrazolium violet (INT) or nitroblue tetrazolium (NBT) together with phenazine methosulfate (PMS) (electron-transfer intermediate), ferricyanide, and horse heart cytochrome c (but not NAD(P)+ or oxygen) were additional artificial electron acceptors. Since other opines were not available commercially, more detailed substrate specificity was alternatively estimated by an inhibition study (Figure 4D). The IC50 value for α-ketoglutarate was ~17-fold higher than that for pyruvate, thereby confirming the preference for nopaline over octopine. On the other hand, among several
2.5. Octopine dehydrogenase from Bradyrhizobium japonicum
Bradyrhizobium japonicum is a nitrogen-fixing bacteria found in the roots of a soybean plant, Glycine max. Interestingly, the homologous gene cluster with odhB-odhC-odhA from P. putida contained an additional odhB gene, in the order of odhB1-odhC-odhA-odhB2 (Figure 2A) In order to estimate the subunit assembly of this protein, (His)6-tagged OdhB1 or OdhB2 was co-expressed together with S-tagged OdhA and OdhC in E. coli cells and purified using Ni2+-affinity chromatography. A western blotting analysis using the anti (His)6-tag and S-tag antibodies revealed that the purified proteins both contained not only each OdhB, but also OdhA and OdhC (referred to as BjOdhAB1C and BjOdhAB2C, respectively) (Figure 4A).
Their absorption spectra were similar to that of PpOpnDH, and FAD and FMN (the molar ratio of ~2) were extracted from (orange-brown) them. On the other hand, the significant dehydrogenase activities were detected only toward octopine, although the specific activity of BjOdhAB1C was ~200-fold lower than that of BjOdhAB2C (Figure 3C). The kcat/Km value for the octopine of BjOdhAB2C (using Cl2Ind; 224 min−1 mM−1) was 178-fold lower than that for the nopaline of PpOpnDH. These results suggested that BjOdhAB2C and BjOdhAB1C both functioned as octopine-specific OpnDH (referred to as BjOpnDH2 and BjOpnDH1, respectively).
2.6. Functional role(s) of iron-sulfur clusters
The γ- and α-subunits of OpnDH contain two different types of [Fe-S]-binding motifs consisting of four cysteine residues: [Fe-S]site 1 and [Fe-S]site 2, respectively (Figure 4B). An electron paramagnetic resonance (EPR) spectrum recorded for the frozen solution of the BjOpnDH2 wild-type enzyme (fully reduced by Na2S2O4) at 40 K was composed of at least three paramagnetic species due to [Fe-S] clusters: species A, B, and C (Figure 3E). Among them, only species A was still detected at 60 K, indicating the assignment to be the fully reduced form of [2Fe-2S]− (therefore, species B and C were derived from the reduced form of [4Fe-4S]3−). On the other hand, when one cysteine residue (Cys61; boxed in Figure 4B) in [Fe-S]site 1 was substituted to serine residue, the C61S mutant protein showed the significant activity, and the paramagnetic species was clearly similar to species A found in the wild-type enzyme (Figure 3E). No expression of C382S mutant in [Fe-S]site 2 was found in E. coli (Figure 4B). Wholly, [Fe-S]site 1 and [Fe-S]site 2 of heteromeric OpnDH bind to the [4Fe-4S] and [2Fe-2S] clusters, respectively (Figure 4A), and the latter is important for structural folding and enzyme catalysis.
2.7. Phylogenetic relationship
Flavin-containing OpnDH (the β-subunit) belongs to the
2.8. Physiological role
The degradation of toxic organic compounds by some P. putida strains has been extensively examined . On the other hand, these bacteria, in addition to B. japonicum, also colonize the rhizosphere of agronomically relevant plants at high population densities; the origin of opines may be from rotting plants and/or plant exudates rather than biosynthesis. In case of endophytes, opines may be also provided without their being exuded. It is reported that a P. putida strain isolated from a commercial nursery catabolized nopaline , thereby conforming to the substrate specificity of PpOpnDH. In contrast to Agrobacterium species, PpOpnDH and BjOpnDH genes are located on the chromosome. Indeed, large numbers of Bradyrhizobium species possess the homologous genes, whereas all the other P. putida strains, except for KT2440, do not. These findings suggest that P. putida KT2440 very recently acquired this ability by horizontal gene transfer (not plasmid transfer).
l-Hydroxyproline in nature
3.2. Involvement in bacterial
In contrast to mammals, bacteria metabolize free T4LHyp to α-ketoglutarate through four enzymatic steps via cis-4-hydroxy-
3.3. Homomeric HypDH
Among T4LHyp gene clusters of P. putida KT2440 and (nitrogen-fixing) Sinorhizobium meliloti 2011, PP_1255 and SM_b20267 genes, annotated as FAD-dependent oxidoreductase and
3.4. Heteromeric HypDH
When compared with P. putida and S. meliloti, T4LHyp gene cluster of Pseudomonas aeruginosa PAO1 contained several additional genes, PA1267 (glycine/
In contrast to homomeric HypDHs, both FAD and FMN (the molar ratio of ~2) were detected as prosthetic groups as well as OpnDH. On the other hand, when each subunit was functionally characterized, only proteins containing the HypB were active (Figure 7D). Only FAD was extracted from HypB (~0.8 mol/mol of protein), while FAD and FMN were extracted from HypBC (and HypBCD). These indicated that the dehydrogenation activity of heteromeric HypDH dependents only to β-subunit, and that the γ-subunit had no effect on the binding of FMN. Since two [Fe-S]-binding sites in the γ- and α-subunits were conserved, this enzyme might also contain [4Fe-4S] and [2Fe-2S] clusters as well as OpnDH (see Section 2.6) (Figure 4A).
3.5. Comparison with archaeal
In spite of their same functions, there is only ~20% of sequence similarity of PpHypDH to PaHypDH (β-subunit) in
3.6. Physiological role
It was believed that HypDH is involved only in T4LHyp but not in proline metabolism. In case of S. meliloti, the hypB mutant continues to grow on T4LHyp, but at half the wild-type rate, which may be due to alternative
In addition to T4LHyp, other relatively rare