An NAD(+)-dependent L-tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH) was cloned and overexpressed in Escherichia coli. The recombinant NpTrpDH with a C-terminal His(6)-tag was purified to homogeneity using a Ni-NTA agarose column, and was found to be a homodimer with a molecular mass of 76.1 kDa. The enzyme required NAD(+) and NADH as cofactors for oxidative deamination and reductive amination, respectively, but not NADP(+) or NADPH. L-Trp was the preferred substrate for deamination, though L-Phe was deaminated at a much lower rate. The enzyme exclusively aminated 3-indolepyruvate; phenylpyruvate was inert. The pH optima for the deamination of L-Trp and amination of 3-indolpyruvate were 11.0 and 7.5, respectively. For deamination of L-Trp, maximum enzymatic activity was observed at 45 degrees C. NpTrpDH retained more than 80% of its activity after incubation for 30 min at pHs ranging from 5.0 to 11.5 or incubation for 10 min at temperatures up to 40 degrees C. Unlike L-Trp dehydrogenases from higher plants, NpTrpDH activity was not activated by metal ions. Typical Michaelis-Menten kinetics were observed for NAD(+) and L-Trp for oxidative deamination, but with reductive amination there was marked substrate inhibition by 3-indolepyruvate. NMR analysis of the hydrogen transfer from the C4 position of the nicotinamide moiety of NADH showed that NpTrpDH has a pro-S(B-type)stereospecificity similar to the Glu/Leu/Phe/Val dehydrogenase family. (C) 2014 Elsevier Inc. All rights reserved.