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

Paper ID Volume ID Publish Year Pages File Format Full-Text
16933 42623 2014 7 PDF Available
Title
Biochemical characterization of an l-tryptophan dehydrogenase from the photoautotrophic cyanobacterium Nostoc punctiforme
Abstract

•A TrpDH gene homolog from Nostoc punctiforme NIES-2108 was cloned and overexpressed.•The product showed strong NAD+-dependent TrpDH activity.•Marked substrate inhibition by 3-indolepyruvate was observed.•NpTrpDH had a homodimeric structure with a molecular mass of 76.1 kDa.•NpTrpDH showed B-type stereospecificity for hydrogen transfer from NADH.

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 76.1 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 °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 °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.

Keywords
TrpDH, l-Trp dehydrogenase; NpTrpDH, NAD+-dependent l-tryptophan dehydrogenase from Nostoc punctiforme NIES-2108l-Tryptophan dehydrogenase; NAD+; Nostoc punctiforme; Amino acid dehydrogenase; B-type stereospecificity
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Biochemical characterization of an l-tryptophan dehydrogenase from the photoautotrophic cyanobacterium Nostoc punctiforme
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 60, 10 June 2014, Pages 40–46
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us