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J. Biol. Chem., Vol. 282, Issue 32, 23687-23697, August 10, 2007

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A Novel Form of 6-Phosphofructokinase

IDENTIFICATION AND FUNCTIONAL RELEVANCE OF A THIRD TYPE OF SUBUNIT IN PICHIA PASTORIS^*

Katrin Tanneberger, Jürgen Kirchberger, Jörg Bär, Wolfgang Schellenberger, Sven Rothemund, Manja Kamprad^¶, Henning Otto^||, Torsten Schöneberg¹, and Anke Edelmann

From the Institute of Biochemistry, Molecular Biochemistry, Medical Faculty, University of Leipzig, Johannisallee 30, 04103 Leipzig, Germany, the Interdisziplinäres Zentrum für Klinische Forschung (IZKF) core unit Peptide Technologies, Medical Faculty, University of Leipzig, Inselstrasse 22, 04103 Leipzig, Germany, the ^¶Institute of Clinical Immunology and Transfusion Medicine, Medical Faculty, University of Leipzig, Johannisallee 30, 04103 Leipzig, Germany, and the ^||Institute of Chemistry and Biochemistry, Free University Berlin, Thielallee 63, 14195 Berlin, Germany

Classically, 6-phosphofructokinases are homo- and hetero-oligomeric enzymes consisting of subunits and / subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, , , and . The sequence of the subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the subunit is a constitutive component of Pfk from Pichia pastoris (PpPfk). Analyses of the purified PpPfk suggest a heterododecameric assembly from the three different subunits. Accordingly, it is the largest and most complex Pfk identified yet. Although, the subunit is not required for enzymatic activity, the subunit-deficient mutant displays a decreased growth on nutrient limitation and reduced cell flocculation when compared with the P. pastoris wild-type strain. Subsequent characterization of purified Pfks from wild-type and subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the subunit. Therefore, we suggest that the subunit contributes to adaptation of P. pastoris to energy resources.

Received for publication, December 18, 2006 , and in revised form, May 23, 2007.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY686600 [GenBank] .

^* This work was supported by the IZKF-Leipzig and the Bundesministerium für Bildung und Forschung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and Figs. S1-S12 and additional references.

¹ To whom correspondence should be addressed. Tel.: 49-341-972-2150; Fax: 49-341-972-2159; E-mail: schoberg{at}medizin.uni-leipzig.de.

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