Structural Insights into l-Tryptophan Dehydrogenase from a Photoautotrophic Cyanobacterium, Nostoc punctiforme.
l-Tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH), despite exhibiting high amino acid sequence identity (>30%)/homology (>50%) with NAD(P)+-dependent l-Glu/l-Leu/l-Phe/l-Val dehydrogenases, exclusively catalyzes reversible oxidative deamination of l-Trp to 3-indolepyruvate in the presence of NAD+Here, we determined the crystal structure of the apo form of NpTrpDH. The structure of the NpTrpDH monomer, which exhibited high similarity to that of l-Glu/l-Leu/l-Phe dehydrogenases, consisted of a substrate-binding domain (domain I, residues 3 to 133 and 328 to 343) and an NAD+/NADH-binding domain (domain II, residues 142 to 327) separated by a deep cleft. The apo-NpTrpDH existed in an open conformation, where domains I and II were apart from each other. The subunits dimerized themselves mainly through interactions between amino acid residues around the β-1 strand of each subunit, as was observed in the case of l-Phe dehydrogenase. The binding site for the substrate l-Trp was predicted by a molecular docking simulation and validated by site-directed mutagenesis. Several hydrophobic residues, which were located in the active site of NpTrpDH and possibly interacted with the side chain of the substrate l-Trp, were arranged similarly to that found in l-Leu/l-Phe dehydrogenases but fairly different from that of an l-Glu dehydrogenase. Our crystal structure revealed that Met-40, Ala-69, Ile-74, Ile-110, Leu-288, Ile-289, and Tyr-292 formed a hydrophobic cluster around the active site. The results of the site-directed mutagenesis experiments suggested that the hydrophobic cluster plays critical roles in protein folding, l-Trp recognition, and catalysis. Our results provide critical information for further characterization and engineering of this enzyme.



In this study, we determined the three-dimensional structure of l-Trp dehydrogenase, analyzed its various site-directed substitution mutants at residues located in the active site, and obtained the following informative results. Several residues in the active site form a hydrophobic cluster, which may be a part of the hydrophobic core essential for protein folding. To our knowledge, there is no previous report demonstrating that a hydrophobic cluster in the active site of any l-amino acid dehydrogenase may have a critical impact on protein folding. Furthermore, our results suggest that this hydrophobic cluster could strictly accommodate l-Trp. These studies show the structural characteristics of l-Trp dehydrogenase and hence would facilitate novel applications of l-Trp dehydrogenase.
Agricultural Science, Graduate School of Integrated Arts and Sciences, Kochi University, Kochi, Japan t-wakamatsu@kochi-u.ac.jp.
Taisuke
T
Wakamatsu
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Kagawa, Japan.
Haruhiko
H
Sakuraba
Agricultural Science, Graduate School of Integrated Arts and Sciences, Kochi University, Kochi, Japan.
Megumi
M
Kitamura
Agricultural Science, Graduate School of Integrated Arts and Sciences, Kochi University, Kochi, Japan.
Yuichi
Y
Hakumai
Department of Biochemistry, Osaka Medical College, Osaka, Japan.
Kenji
K
Fukui
Research Institute of Molecular Genetics, Kochi University, Kochi, Japan.
Kouhei
K
Ohnishi
Agricultural Science, Graduate School of Integrated Arts and Sciences, Kochi University, Kochi, Japan.
Makoto
M
Ashiuchi
Department of Biomedical Engineering, Osaka Institute of Technology, Osaka, Japan.
Toshihisa
T
Ohshima
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D003460:Crystallization; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D046937:Nostoc / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment
D001426:Bacterial Proteins; D010088:Oxidoreductases
crystal structure; folding; hydrophobic cluster; l-tryptophan dehydrogenase; substrate recognition