Photon Factory Activity Report 2018 #36 (2019)

AR-NE3A/2017G010

Crystal structure of L-tryptophan dehydrogenase
Taisuke Wakamatsu 1, *, Haruhiko Sakuraba 2, *, Megumi Kitamura 1, Yuichi Hakumai1, Kenji Fukui
3, Kouhei Ohnishi 4, Makoto Ashiuchi 1, and Toshihisa Ohshima 5
1
Agricultural Science, Graduate School of Integrated Arts and Sciences, Kochi University, Kochi,Japan
2
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Kagawa,Japan
3
Department of Biochemistry, Osaka Medical College, Osaka, Japan
4
Research Institute of Molecular Genetics, Kochi University, Kochi, Japan
5
Department of Biomedical Engineering, Osaka Institute of Technology, Osaka, Japan

1 Introduction
NAD(P)-dependent L-amino acid dehydrogenases (EC1.4.1.x) catalyze reversible oxidative deamination of Lamino acids to their corresponding 2-oxo acids andammonia in the presence of NAD(P). So far, more thanfifteen types of L-amino acid dehydrogenases have been
identified from various organisms and characterized
extensively. In particular, detailed structure and function
analyses of L-Glu/L-Leu/L-Phe dehydrogenases have led tothe elucidation of their catalytic mechanisms.
Consequently, several L-amino acid dehydrogenases havebeen successfully used for the syntheses for chiral aminoacids and their analogs, for developing biosensors for Lamino acids.
Among all the amino acid dehydrogenases, biochemical
information and application on the L-Trp dehydrogenase(TrpDH, EC 1.4.1.19) have been limited so far, mainly due
to its extremely limited distribution and less stability. The
enzyme is known to catalyze reversible oxidative
deamination of L-Trp to 3-indolepyruvate (IPyA) in thepresence of NAD(P). IPyA is known to show
pharmacological and antioxidant effects. TrpDH has highpotentiality for applications such as synthesis of IPyA andbiosensor for L-Trp/IPyA. The enzyme was first identified
in several higher plants, including Pisum sativum, in the
middle of 1980s, and was partially characterized at thattime. Since then, no studies were reported for this enzyme
until Npun_R1275 from a cyanobacterium Nostoc
punctiforme ATCC 29133 (29133TrpDH) was identified tobe the first NAD-dependent TrpDH of microbial origin.
We recently characterized TrpDH from N. punctiforme
NIES-2108 (NpTrpDH), which is a 29133TrpDH
homologous protein and found that the enzyme catalyzed
reversible oxidative deamination of L-Trp to IPyA in thepresence of NAD and exhibited B-type stereospecificity
for transferring hydrogen from NADH, just like L-Glu/LLeu/L-Phe/L-Val dehydrogenases. The most striking
feature was that NpTrpDH uses L-Trp as the preferred
electron donor for oxidative deamination and exhibits
much less activity when L-Phe was used as the electrondonor. Other proteinogenic L-amino acids and D-Trp, thestereoisomer of L-Trp, were all inert as electron donors forNpTrpDH. For reductive amination, IPyA was the mostpreferred substrate, whereas phenylpyruvate, the 2-oxo
analog of L-Phe, was inert as a substrate. Despite theseinteresting observations, there exists no structural
information on TrpDH. Thus, we employed X-ray
crystallography to solve the apo-structure of NpTrpDH,
2 Experiment
Data were collected under cryo conditions at theBeamline AR-NE3A at Photon Factory in Japan. The
crystal structure of LeuDH from Sporosarcina
psychrophila (SpLeuDH, PDB ID: 3VPX, amino acidsequence identity: 49%) was applied as a search model,
and the program PHENIX was used for molecularreplacement phase determination and initial modelbuilding. Model building was performed using theprogram Coot, and refinement was carried out using
Refmac5.

Fig. 1: Overall structure of NpTrpDH.
3 Results and Discussion
The structure of NpTrpDH monomer, which exhibitedhigh similarity to the structures of L-Glu/L-Leu/L-Phe
dehydrogenases, consisted of a substrate-binding domain(domain I: residues: 2–133 and 328–343) and a Rossmannfold domain (domain II, residues: 142–327) separated by adeep cleft. The apo-NpTrpDH existed in an open
conformation, where the domains I and II move apart fromeach other. The subunits dimerized themselves mainly due
to interactions between amino acid residues around the 1 strand of each subunit (Fig. 1), as was observed in thecase of L-Phe dehydrogenase. Several hydrophobic

Photon Factory Activity Report 2018 #36 (2019)residues, which were located in the active site of NpTrpDHand possibly interacted with the side-chain of the substrate
L-Trp, were arranged similar to those found in L-Leu/L-Phe
dehydrogenases.
To predict the possible L-Trp binding site, we initiallyperformed a molecular docking using the MOE software
tool. The calculated binding energy was -14 kcal/mol,which suggested that this binding model was reasonablesignificantly. In contrast, the calculated binding energy
with L-Phe was 0.18 kcal/mol, which indicated that L-Phe
is unsuitable as a substrate compared with L-Trp. In thismodel, the side-chain of L-Trp was hydrophobicallyinteracted with those of Ala-42 (4.1Å), Met-65 (3.3Å),Lys-68 (4.2Å), Gly-112 (4.0Å), Leu-288 (3.7Å), and Val291 (3.7Å) (Fig. 2). Based on these results and active sites
comparison, we identified several residues including otherhydrophobic residues around the active site as thecandidate residues which might be involved in recognizing
the side chain of L-Trp.

Fig. 2: Predicted L-Trp-binding site in the NpTrpDH.
The docking simulation was performed using the MOE
software tool.
The site-directed substitution mutants at residues Met40, Met-65, Ala-69, Ile-74, Ile-110, Val-132, and Val-133
in domain I and Leu-288, Ile-289, Val-291, Tyr-292, Met295, and Ile-296 in domain II of NpTrpDH revealed that
Met-40, Ala-69, Ile-74, Ile-110, Leu-288, Ile-289, and Tyr292 formed a hydrophobic cluster, which may play a
critical role in both protein folding and L-Trp recognition.
To our knowledge, there is no previous reportdemonstrating that a hydrophobic cluster in the active siteof any L-amino acid dehydrogenase may have a critical
impact on protein folding. Furthermore, our results suggestthat this hydrophobic cluster could strictly andstereoselectively accommodate L-Trp as compared to otherL-amino acid dehydrogenases such as L-Glu/L-Leu/L-Phedehydrogenases. These studies not only show the firststructural characteristics of L-Trp dehydrogenase, but may
also facilitate the novel applications of L-Trpdehydrogenase.
Acknowledgement
We are grateful to the staff of the Photon Factory for
their assistance with data collection, which was approvedby the Photon Factory Program Advisory Committee.

Reference[1] T. Wakamatsu et al., Appl. Env. Microbiol. e02710-16(2017).
* t-wakamatsu@kochi-u.ac.jp* sakuraba@ag.kagawa-u.ac.jp