TKL2, a second transketolase gene of Saccharomyces cerevisiae

Cloning, sequence and deletion analysis of the gene

Authors

  • Ine SCHAAFF-GERSTENSCHLÄGER,

    Corresponding author
    1. Institut für Mikrobiologie, Technische Hochschule Darmstadt, Germany
      Correspondence to I. Schaaff-Gerstenschläger, Institut für Mikrobiologie, Technische Hochschule Darmstadt, Schnittspahnstrasse 10, D-64287 Darmstadt, Germany
      Fax: +49 6151 164808.
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  • Gertrud MANNHAUPT,

    1. Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie, Ludwig-Maximilians-Universität München, Germany
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  • Irene VETTER,

    1. Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie, Ludwig-Maximilians-Universität München, Germany
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  • Friedrich K. ZIMMERMANN,

    1. Institut für Mikrobiologie, Technische Hochschule Darmstadt, Germany
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  • Horst FELDMANN

    1. Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie, Ludwig-Maximilians-Universität München, Germany
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  • Note. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data banks and are available under accession number X73532.

Correspondence to I. Schaaff-Gerstenschläger, Institut für Mikrobiologie, Technische Hochschule Darmstadt, Schnittspahnstrasse 10, D-64287 Darmstadt, Germany
Fax: +49 6151 164808.

Abstract

Transketolase activity is indispensable for the generation of erythrose 4-phosphate and therefore necessary for the biosynthesis of the aromatic amino acids. Yeast mutants with a deletion of the transketolase gene, TKL1, can grow without aromatic amino acid supplement indicating an additional source of erythrose 4-phosphate in the cells. Here we describe the cloning of TKL2, a gene coding for a second transketolase enzyme in Saccharomyces cerevisiae. The deduced protein sequence of TKL2 demonstrates 71% identitity with TKL1 [Sundström, M., Lindqvist, Y., Schneider, G., Hellman, U. & Ronne, H. (1993) J. Biol. Chem., in the press]. Double mutants for both genes, TKL1 and TKL2, are auxotrophic for aromatic amino acids, indicating a complete block in the transketolase activity. Deletion of TKL2 alone does not lead to a significant phenotype, and transketolase activity is not reduced in these mutants. Overexpression of TKL2 on a multi-copy plasmid in a tkl1 background showed that TKL2 is functionally expressed: transketolase enzyme activity was detectable in the transformants and the protein reacts with anti-transketolase serum in Western blot analysis. In addition, transformation of the tkl1 tkl2 double mutant with the TKL2 plasmid can compensate the growth defect on a medium without aromatic amino acids.

Enzymes
 

Formaldehyde transketolase (EC 2.2.1.3)

 

glucose 6-phosphate 1-dehydrogenase (EC 1.1.1.49)

 

glycerol-3-phosphate dehydrogenase (EC 1.1.99.5)

 

transketolase (EC 2.2.1.1)

 

triose-phosphate isomerase (EC 5.3.1.1)

 

xylose isomerase (EC 5.3.1.5)

 

xylulokinase (EC 2.7.1.17)

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