Structural insights into Ltryptophan dehydrogenase from a photoautotrophic cyanobacterium, Nostoc punctiforme

Abstract

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.