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The intramolecular dehydro-Diels–Alder reaction of 1-indolyl-1,6-heptadiynes proceeds smoothly under rather mild heating conditions to give substituted carbazoles in moderate to good yields. The reaction of 7-aryl-1-indolyl-1,6-heptadiynes under heating gives the corresponding carbazoles chemoselectively in high yields, whereas the reaction under basic conditions gives naphthalenes as major products.

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. IMPORTANCE 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.

Little is known about an enhanced lattice defect formation due to an interaction between hydrogen and dislocation in face-centered cubic (fcc) metals such as stainless steels. In the present study, hydrogen spectra evolved from Type 316L and 304 stainless steels during elastic and plastic deformation were detected using a quadrupole mass spectrometer. The amount of lattice defect enhanced by hydrogen and strain was measured using thermal desorption analysis. For 316L stainless steel, hydrogen desorption increased rapidly when plastic deformation began, since the dislocation dragged hydrogen to the surface of the specimen. In contrast, hydrogen desorption increased with applying strain for 304 stainless steel, because of phase transformation from austenite into martensite with larger hydrogen diffusivity. And the amount of desorbed hydrogen increased with decreasing strain rate. These results indicate that dislocation can drag and transport large amounts of hydrogen when the dislocation velocity approaches the hydrogen diffusion rate. The amount of lattice defects in stainless steels was enhanced by hydrogen and applied strain. The most probable reason for the increase in the amount of lattice defects can be ascribed to the increase in the amount of vacancy clusters. These findings lead to the conclusion that the interaction between dislocation and hydrogen enhances the formation of vacancy clusters, as a result, causes hydrogen embrittlement.Copyright © 2013 by ASME