5b37
Crystal structure of L-tryptophan dehydrogenase from Nostoc punctiforme
Structural highlights
FunctionTRPDH_NOSPU Catalyzes the reversible oxidative deamination of L-tryptophan to indole-3-pyruvate in the presence of NAD(+) (PubMed:24835098, PubMed:27815281). Shows weak activity with L-phenylalanine, but cannot use other L-amino acids and D-Trp (PubMed:24835098, PubMed:27815281). Cannot use NADP(+) for oxidative deamination of L-Trp, and shows only weak activity with NADPH for reductive amination of indole-3-pyruvate (PubMed:24835098). Involved in the biosynthesis of scytonemin, a cyanobacterial radiation-absorbing pigment (Probable).[1] [2] [3] Publication Abstract from PubMedL-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 apo-form of NpTrpDH. The structure of NpTrpDH monomer, which exhibited high similarity to those of L-Glu/L-Leu/L-Phe dehydrogenases, consisted of a substrate-binding domain (domain I, residues: 3-133 and 328-343) and an NAD+/NADH-binding domain (domain II, residues: 142-327) separated by a deep cleft. The apo-NpTrpDH existed in an open conformation, where the domains I and II were apart from each other. The subunits dimerized themselves mainly through interactions between amino acid residues around the beta-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 mutageneses. 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 those found in L-Leu/L-Phe dehydrogenases but fairly different from that of a 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. Results of site-directed mutagenesis experiments suggested that the hydrophobic cluster plays critical roles in protein folding, L-Trp recognition, and catalysis. Our results would provide critical information for further characterization and engineering of this enzyme. IMPORTANCE STATEMENT: In this study, we determined the first 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 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 first structural characteristics of L-Trp dehydrogenase, and hence, would facilitate the novel applications of L-Trp dehydrogenase. Structural insights into L-tryptophan dehydrogenase from a photoautotrophic cyanobacterium Nostoc punctiforme.,Wakamatsu T, Sakuraba H, Kitamura M, Hakumai Y, Fukui K, Ohnishi K, Ashiuchi M, Ohshima T Appl Environ Microbiol. 2016 Nov 4. pii: AEM.02710-16. PMID:27815281[4] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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