Understanding the catalytic mechanism of flavin-containing
dehydrogenases and their bioindustrial applications
Seiya WATANABE
Department of Bioscience, Graduate School of Agriculture,Ehime University
Research objective
Octopine and nopaline are synthesized in crown gall tumor tissues by NAD(P)H-dependent
soluble dehydrogenases from pathogenic Agrobacterium tumefaciens, which catalyze thereductive condensation of pyruvate (for octopine) or α-ketoglutarate (for nopaline) with
L-arginine. This bacterium possesses membrane-associated dehydrogenase(s), by which
opines are utilized as selective growth substrates that favor their propagation. Therefore, it ispossible that the characterization of this enzyme will lead to the generation of agriculturalchemicals. Previously, I biochemically characterized homologous proteins from thenon-pathogenic bacteria Pseudomonas putida KT2440 and Bradyrhizobium japonicum
USDA110 at the first time. The novel opine dehydrogenase (Odh) 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 are commonly contained as aprosthetic group(s). This enzyme has high potential for utilization as a specific element forelectrochemical processing (e.g., in enzyme sensors and batteries) because electrons from thesubstrate can be introduced to an electrode using an artificial dye as the mediator. Based onthese insights, I aimed to determine the crystal structure of putative flavin-dependent Odh
from Aureimonas altamirensis by elucidating the catalytic mechanism at the atomic level.
Methods
1. Expression and purification of AaOdbB3 gene
The putative opine dehydrogenase gene from A. altamirensis (AUL31_RS11475; AaOdhB3)
was cloned into a pACYC-Duet vector. Recombinant (His)6-tagged AaOdhB3 protein wasoverexpressed by isopropyl-β-D-thiogalactopyranoside (IPTG) in Escherichia coli
BL21(DE3) cells and purified using Ni-NTA affinity chromatography.
2. Functional characterization
To estimate potential opine dehydrogenase activity, AaOdhB3 was incubated with nopalineor octopine in the presence of p-iodonitrotetrazolium violet (INT) and phenazine methylsulfate (PMS) as an artificial electron acceptor and mediator, respectively. The yield ofL-arginine was measured using an amino acid analyzer.
3. Preliminary crystallization
All crystallization trials were performed at 20 °C using the sitting-drop vapor-diffusion
method. Drops (0.5 μL) of ~16 mg/ml AaOdhB3 protein were mixed with equal amounts ofreservoir solution and equilibrated against 70 μL of the same reservoir solution by vapor
diffusion. The initial trial was performed using Index HT and Crystal Screen (HamptonResearch). The best crystal of AaOdhB3 was obtained with Index HT A4, C3, G2, andCrystal Screen HT B3, F10, G2, and H2 conditions.
4. Optimization of crystallization conditions
Among several crystallization conditions, Index HT G2 (0.2 M lithium sulfate monohydrate,
0.1 M BIS-TRIS (pH 5.5), 25% (w/v) PEG 3350) was further optimized.
5. Data collection of native crystals
Diffraction data of native crystals of AaOdhB3 were collected using the PILATUS 6M
detector of BL45XU at SPring-8 (Hyogo, Japan). Although the resolution was significantly
high (2Å>), it was unsuccessful in determining the structure, possibly due to the low
sequence identity with the model structure for the molecular replication method.
6. Determination of the structure using Se-Met derivative protein
Alternatively, the crystal structure was elucidated by the single-wavelength anomalous
dispersion (SAD) method using selenomethionine (SeMet)-substituted crystal andsubsequently refined to a resolution of 1.5 Å against the data set from the native crystal.
7. Site-directed mutagenic analysis
Based on this structure, several alanine mutants of amino acids close to the putative active
pocket were designed and their activity elucidated.
ResultsFigure 1. Crystals for native (left) and SeMet-substituted forms (right)

All crystals of AaOdhB3 were yellow, suggesting the binding of flavin nucleotides. Thecrystal structure was determined at a 1.5 Å resolution by the SAD method using aselenomethionine (SeMet)-substituted crystal. AaOdhB3 is a homodimer and each monomer
contains a bidomain architecture composed of a FAD-binding domain and asubstrate-binding domain. The overall structural folding of AaOdhB3 was similar to those offlavin-dependent dehydrogenases (oxidases), including glycine oxidase, D-arginine
dehydrogenase, and L-glutamate oxidase. On the other hand, there was low sequencesimilarity (~30%) between AaOdhB3 and them. FAD binds the enzyme tightly by interactingwith Ile11, Trhr38, Ser39, Trp45, Val178, Val340, and Thr341 within the FAD-binding
domain (Fig. 2, right panel). Among the two chains A and B, only chain A contained
polyethylene glycol in the putative active pocket, derived from the crystallization conditions(Fig. 3). Superimposition of the two chains revealed that the side chains of Tyr57, His337,and Arg313 in chain A appeared to undergo significant appears conformational changes
compared to chain B, and the loop between β3 and α6 was partially disordered.

Figure 2．Overall structure (left) and FAD binding site (right)

Among the commercially available nopaline and octopine, AaOdhB3 only catalyzed thedehydrogenation reaction of nopaline to produce α-ketoglutarate and L-arginine. However, it
was not possible to obtain crystals in complexes with these compounds. It is likely that theconformational difference between chains A and B partially mimics substrate binding.
Therefore, we selected the 17 amino acid residues close to putative active site, which weresubstituted to alanine. Among them, W45A, R312A, H337A, S338A, and T341A werecompletely inactive, and E50A, Y57A, R247A, and P315A dramatically decreased theactivity. Since a conformational change was observed in the above underlined residues, it islikely that some of them actually play a role as active sites. Furthermore, these residues arefound only in AaOdhB3 but not in other enzymes belonging to the same protein superfamily,indicating the importance of these residues in recognizing opine compounds with complex
structures.

Conclusion
In this study, the crystal structure of flavin-dependent opine dehydrogenase was determined
for the first time. However, there are two possibilities for trace activity toward nopaline. First,
nopaline is not the best substrate for AaOdhB3. Opine is synthesized by the reductive
condensation of α-keto acid and amino acids. α-Ketoglutarate is necessary for AaOdhB3 asan α-keto acid, whereas L-arginine may be not usually best as an amino acid. Second,
AaOdhB3 corresponds to a catalytic subunit of a heteromeric enzyme. I am currently
working on elucidating these hypotheses.

Figure 3．Conformational change between chains A and B (left) and PEG binding (right)

References
None.