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

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

研究成果: Contribution to journalArticle査読

3 被引用数 (Scopus)

抄録

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.

本文言語英語
ページ(範囲)40-46
ページ数7
ジャーナルEnzyme and Microbial Technology
60
DOI
出版ステータス出版済み - 6 10 2014

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology

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