Biochemical characterization of an L-tryptophan dehydrogenase from the photoautotrophic cyanobacterium Nostoc punctiforme

Ryutaro Ogura, Taisuke Wakamatsu, Yuta Mutaguchi, Katsumi Doi, Toshihisa Ohshima

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

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 His6-tag was purified to homogeneity using a Ni-NTA agarose column, and was found to be a homodimer with a molecular mass of 76.1kDa. 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°C. NpTrpDH retained more than 80% of its activity after incubation for 30min at pHs ranging from 5.0 to 11.5 or incubation for 10min at temperatures up to 40°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.

Original languageEnglish
Pages (from-to)40-46
Number of pages7
JournalEnzyme and Microbial Technology
Volume60
DOIs
Publication statusPublished - Jun 10 2014

Fingerprint

Nostoc
Amination
Deamination
Cyanobacteria
Tryptophan
NAD
Oxidoreductases
Enzymes
His-His-His-His-His-His
NADP
Molecular mass
Substrates
Escherichia coli
Metal ions
Nuclear magnetic resonance
Niacinamide
Hydrogen
Kinetics
Sepharose
Metals

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology

Cite this

Biochemical characterization of an L-tryptophan dehydrogenase from the photoautotrophic cyanobacterium Nostoc punctiforme. / Ogura, Ryutaro; Wakamatsu, Taisuke; Mutaguchi, Yuta; Doi, Katsumi; Ohshima, Toshihisa.

In: Enzyme and Microbial Technology, Vol. 60, 10.06.2014, p. 40-46.

Research output: Contribution to journalArticle

Ogura, Ryutaro ; Wakamatsu, Taisuke ; Mutaguchi, Yuta ; Doi, Katsumi ; Ohshima, Toshihisa. / Biochemical characterization of an L-tryptophan dehydrogenase from the photoautotrophic cyanobacterium Nostoc punctiforme. In: Enzyme and Microbial Technology. 2014 ; Vol. 60. pp. 40-46.
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N2 - 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 His6-tag was purified to homogeneity using a Ni-NTA agarose column, and was found to be a homodimer with a molecular mass of 76.1kDa. 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°C. NpTrpDH retained more than 80% of its activity after incubation for 30min at pHs ranging from 5.0 to 11.5 or incubation for 10min at temperatures up to 40°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.

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