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Journal of Bacteriology, August 2000, p. 4188-4197, Vol. 182, No. 15
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

HARO7 Encodes Chorismate Mutase of the Methylotrophic Yeast Hansenula polymorpha and Is Derepressed upon Methanol Utilization

Sven Krappmann,1 Ralph Pries,1 Gerd Gellissen,2 Mark Hiller,3 and Gerhard H. Braus1,*

Institute of Microbiology and Genetics, Georg August University, D-37077 Göttingen,1 Rhein Biotech GmbH, D-40595 Düsseldorf,2 and Institute of Microbiology, Heinrich Heine University, D-40225 Düsseldorf,3 Germany

Received 10 April 2000/Accepted 16 May 2000

The HARO7 gene of the methylotrophic, thermotolerant yeast Hansenula polymorpha was cloned by functional complementation. HARO7 encodes a monofunctional 280-amino-acid protein with chorismate mutase (EC 5.4.99.5) activity that catalyzes the conversion of chorismate to prephenate, a key step in the biosynthesis of aromatic amino acids. The HARO7 gene product shows strong similarities to primary sequences of known eukaryotic chorismate mutase enzymes. After homologous overexpression and purification of the 32-kDa protein, its kinetic parameters (kcat = 319.1 s-1, nH = 1.56, [S]0.5 = 16.7 mM) as well as its allosteric regulatory properties were determined. Tryptophan acts as heterotropic positive effector; tyrosine is a negative-acting, heterotropic feedback inhibitor of enzyme activity. The influence of temperature on catalytic turnover and the thermal stability of the enzyme were determined and compared to features of the chorismate mutase enzyme of Saccharomyces cerevisiae. Using the Cre-loxP recombination system, we constructed mutant strains carrying a disrupted HARO7 gene that showed tyrosine auxotrophy and severe growth defects. The amount of the 0.9-kb HARO7 mRNA is independent of amino acid starvation conditions but increases twofold in the presence of methanol as the sole carbon source, implying a catabolite repression system acting on HARO7 expression.

* Corresponding author. Mailing address: Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstr. 8, D-37077 Göttingen, Germany. Phone: (49) (0)551/39-3770. Fax: (49) (0)551/39-3820. E-mail: gbraus{at}gwdg.de.

Journal of Bacteriology, August 2000, p. 4188-4197, Vol. 182, No. 15
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Helmstaedt, K., Heinrich, G., Lipscomb, W. N., Braus, G. H. (2002). Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal chorismate mutase. Proc. Natl. Acad. Sci. USA 99: 6631-6636 [Abstract] [Full Text]  
  • Helmstaedt, K., Krappmann, S., Braus, G. H. (2001). Allosteric Regulation of Catalytic Activity: Escherichia coli Aspartate Transcarbamoylase versus Yeast Chorismate Mutase. Microbiol. Mol. Biol. Rev. 65: 404-421 [Abstract] [Full Text]  
  • Krappmann, S., Lipscomb, W. N., Braus, G. H. (2000). Coevolution of transcriptional and allosteric regulation at the chorismate metabolic branch point of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 10.1073/pnas.240469697v1 [Abstract] [Full Text]  
  • Krappmann, S., Lipscomb, W. N., Braus, G. H. (2000). Coevolution of transcriptional and allosteric regulation at the chorismate metabolic branch point of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 97: 13585-13590 [Abstract] [Full Text]  


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