thermostable alanine racemase of bacillus stearothermophilus construction and expression of active fragmentary enzyme

Hirohide Toyama, Katsuyuki Tanizawa, Tohru Yoshimura, Shigehiro Asano, Young Hee Lim, Nobuyoshi Esaki, Kenji Soda

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Limited proteolysis studies on alanine racemase suggested that the enzyme subunit is composed of two domains (Galakatos, N. G., and Walsh, C. T. (1987) Biochemistry 26, 8475-8480). We have constructed a mutant gene that tandemly encodes the two polypeptides of the Bacillus stearothermophilus enzyme subunit cleaved at the position corresponding to the predicted hinge region. The mutant gene product purified was shown to be composed of two sets of the two polypeptide fragments and was immunologically identical to the wild-type enzyme. The mutant enzyme, i.e. the fragmentary alanine racemase, was active in both directions of the racemization of alanine. The maximum velocity (Vmax) was about half that of the wild-type enzyme, and the Km value was about double. Absorption and circular dichroism spectra of the fragmentary enzyme were similar to those of the wild-type enzyme. An attempt was made to separately express in Escherichia coli a single polypeptide corresponding to each domain, but no protein reactive with the antibody against the wild-type alanine racemase was produced. Therefore, it is suggested that the two polypeptide fragments can fold into an active structure only when they are co-translated and that they correspond to structural folding units in the parental polypeptide chain.

Original languageEnglish
Pages (from-to)13634-13639
Number of pages6
JournalJournal of Biological Chemistry
Volume266
Issue number21
Publication statusPublished - 1991 Dec 1
Externally publishedYes

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Alanine Racemase
Geobacillus stearothermophilus
Bacilli
Enzymes
Peptides
Genes
Proteolysis
Biochemistry
Hinges
Circular Dichroism
Alanine
Escherichia coli
Antibodies

ASJC Scopus subject areas

  • Biochemistry

Cite this

Toyama, H., Tanizawa, K., Yoshimura, T., Asano, S., Lim, Y. H., Esaki, N., & Soda, K. (1991). thermostable alanine racemase of bacillus stearothermophilus construction and expression of active fragmentary enzyme. Journal of Biological Chemistry, 266(21), 13634-13639.

thermostable alanine racemase of bacillus stearothermophilus construction and expression of active fragmentary enzyme. / Toyama, Hirohide; Tanizawa, Katsuyuki; Yoshimura, Tohru; Asano, Shigehiro; Lim, Young Hee; Esaki, Nobuyoshi; Soda, Kenji.

In: Journal of Biological Chemistry, Vol. 266, No. 21, 01.12.1991, p. 13634-13639.

Research output: Contribution to journalArticle

Toyama, H, Tanizawa, K, Yoshimura, T, Asano, S, Lim, YH, Esaki, N & Soda, K 1991, 'thermostable alanine racemase of bacillus stearothermophilus construction and expression of active fragmentary enzyme', Journal of Biological Chemistry, vol. 266, no. 21, pp. 13634-13639.
Toyama, Hirohide ; Tanizawa, Katsuyuki ; Yoshimura, Tohru ; Asano, Shigehiro ; Lim, Young Hee ; Esaki, Nobuyoshi ; Soda, Kenji. / thermostable alanine racemase of bacillus stearothermophilus construction and expression of active fragmentary enzyme. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 21. pp. 13634-13639.
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