Characterization and application of a novel nicotinamide mononucleotide adenylyltransferase from Thermus thermophilus HB8

Kenji Konishi, Shigeru Ueda, Miki Kawano, Susumu Osawa, Tomohiro Tamura, Eisaku Hokazono, Yuzo Kayamori, Shin ichi Sakasegawa

研究成果: ジャーナルへの寄稿記事

1 引用 (Scopus)

抄録

Herein, we describe a novel enzymatic cycling method to measure nicotinamide mononucleotide (NMN) or nicotinic acid mononucleotide (NaMN), which are precursors of NAD biosynthesis. A gene encoding an NMN adenylyltransferase (NMNAT, EC 2.7.7.1) homologue was identified in Thermus thermophilus HB8. The gene from T. thermophilus (TtNMNAT) was engineered for expression in Escherichia coli and the recombinant enzyme found to be stable, retaining full activity after incubation for 45 min at 70°C. The Km values for NMN and ATP were calculated to be 0.263 and 1.27 mM, respectively, with a Vmax value of 60.3 μmoL/min/mg. TtNMNAT was successfully applied to the colorimetric NMN or NaMN assays, which employed (i) adenylation of NMN to NAD by TtNMNAT or adenylation of NaMN to deamido-NAD (NaAD) by TtNMNAT followed by amidation of NaAD to NAD by NAD synthetase (NADS, EC 6.3.1.5) and (ii) an NAD cycling reaction using 12α-hydroxysteroid dehydrogenase (12α-HSD, EC 1.1.1.176) and diaphorase (DI, EC 1.6.99.3) to accumulate reduced WST-8. This enzymatic cycling method enabled detection of 0.5 μM (12.2 nM in the reaction mixture) NMN or NaMN in an automatic clinical analyzer.

元の言語英語
ページ(範囲)385-389
ページ数5
ジャーナルJournal of Bioscience and Bioengineering
125
発行部数4
DOI
出版物ステータス出版済み - 4 2018

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Nicotinamide-Nucleotide Adenylyltransferase
Nicotinamide Mononucleotide
Thermus thermophilus
NAD
Acids
12 alpha-hydroxysteroid dehydrogenase
Gene encoding
Adenosinetriphosphate
Biosynthesis
Hydroxysteroid Dehydrogenases
Escherichia coli
Assays
Enzymes
Genes
Adenosine Triphosphate
nicotinate mononucleotide

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

これを引用

Characterization and application of a novel nicotinamide mononucleotide adenylyltransferase from Thermus thermophilus HB8. / Konishi, Kenji; Ueda, Shigeru; Kawano, Miki; Osawa, Susumu; Tamura, Tomohiro; Hokazono, Eisaku; Kayamori, Yuzo; Sakasegawa, Shin ichi.

:: Journal of Bioscience and Bioengineering, 巻 125, 番号 4, 04.2018, p. 385-389.

研究成果: ジャーナルへの寄稿記事

Konishi, Kenji ; Ueda, Shigeru ; Kawano, Miki ; Osawa, Susumu ; Tamura, Tomohiro ; Hokazono, Eisaku ; Kayamori, Yuzo ; Sakasegawa, Shin ichi. / Characterization and application of a novel nicotinamide mononucleotide adenylyltransferase from Thermus thermophilus HB8. :: Journal of Bioscience and Bioengineering. 2018 ; 巻 125, 番号 4. pp. 385-389.
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AU - Konishi, Kenji

AU - Ueda, Shigeru

AU - Kawano, Miki

AU - Osawa, Susumu

AU - Tamura, Tomohiro

AU - Hokazono, Eisaku

AU - Kayamori, Yuzo

AU - Sakasegawa, Shin ichi

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