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Cloning and characterization of a novel fold-type I branched-chain amino acid aminotransferase from the hyperthermophilic archaeon Thermococcus sp. CKU-1

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Abstract

We successfully cloned a novel branched-chain amino acid aminotransferase (Ts-BcAT; EC 2.6.1.42) gene from the Thermococcus sp. CKU-1 genome and expressed it in the soluble fraction of Escherichia coli Rosetta (DE3) cells. Ts-BcAT is a homodimer with an apparent molecular mass of approximately 92 kDa. The primary structure of Ts-BcAT showed high homology with the fold-type I, subgroup I aminotransferases, but showed little homology with BcATs known to date, i.e., those of Escherichia coli and Salmonella typhimurium, which belong to the fold-type IV, subgroup III aminotransferases. The maximum enzyme activity of Ts-BcAT was detected at 95 °C, and Ts-BcAT did not lose any enzyme activity, even after incubation at 90 °C for 5 h. Ts-BcAT was active in the pH range from 4.0 to 11.0, the optimum pH was 9.5, and the enzyme was stable between pH 6 and 7. The exceptionally low pK a of the nitrogen atom in the Lys258 ε-amino group in the internal aldimine bond of Ts-BcAT was determined to be 5.52 ± 0.05. Ts-BcAT used 21 natural and unnatural amino acids as a substrate in the overall transamination reaction. l-Leucine and other aliphatic amino acids are efficient substrates, while polar amino acids except glutamate were weak substrates. Phylogenetic analysis revealed that Ts-BcAT is a novel fold-type I, subgroup I branched-chain aminotransferase.

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Abbreviations

AspAT:

Aspartate aminotransferase

BcAT:

Branched-chain amino acid aminotransferase

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

CHES:

2-(Cyclohexylamino)ethanesulfonic acid

MES:

2-Morpholinoethanesulfonic acid

PLP:

Pyridoxal 5′-phosphate

PMP:

Pyridoxamine 5′-phosphate

TAPS:

3-(Tris(hydroxymethyl)methyl)aminopropanesulfonic acid

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Acknowledgments

This study was supported in part by a grant from the Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT).

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Authors and Affiliations

  1. Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-Cho, Suita, Osaka-Fu, 564-8680, Japan

    Yuki Uchida, Tsubasa Washio, Ryo Yamasaki & Tadao Oikawa

  2. Organization for Research and Development of Innovative Science and Technology, Kansai University, 3-3-35 Yamate-Cho, Suita, Osaka-Fu, 564-8680, Japan

    Shiro Kato

  3. Department of Chemistry, Osaka Medical College, Takatsuki, Osaka-Fu, 569-8686, Japan

    Hideyuki Hayashi

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  1. Yuki Uchida
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  2. Hideyuki Hayashi
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  3. Tsubasa Washio
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  5. Shiro Kato
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  6. Tadao Oikawa
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Correspondence to Tadao Oikawa.

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Communicated by A. Driessen.

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Uchida, Y., Hayashi, H., Washio, T. et al. Cloning and characterization of a novel fold-type I branched-chain amino acid aminotransferase from the hyperthermophilic archaeon Thermococcus sp. CKU-1. Extremophiles 18, 589–602 (2014). https://doi.org/10.1007/s00792-014-0642-0

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