Jörg Bär

The 10.8-A structure of Saccharomyces cerevisiae phosphofructokinase determined by cryoelectron microscopy: localization of the putative fructose 6-phosphate binding sites.

Phosphofructokinase plays a key role in the regulation of the glycolytic pathway and is responsible for the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate. Although the structure of the bacterial enzyme is well understood, the knowledge is still quite limited for higher organisms given the larger size and complexity of the eukaryotic enzymes. We have studied phosphofructokinase from Saccharomyces cerevisiae in the presence of fructose 6-phosphate by cryoelectron microscopy and image analysis of single particles and obtained the structure at 10.8A resolution. This was achieved by optimizing the illumination conditions to obtain routinely 8-A data from hydrated samples in an electron microscope equipped with an LaB(6) and by improving the image alignment techniques. The analysis of the structure has evidenced that the homology of the subunits at the sequence level has transcended to the structural level. By fitting the X-ray structure of the bacterial tetramer into each dimer of the yeast octamer the putative binding sites for fructose 6-phosphate were revealed. The data presented here in combination with molecular replacement techniques have served to provide the initial phases to solve the X-ray structure of the yeast phosphofructokinase.

V2 vasopressin receptor deficiency causes changes in expression and function of renal and hypothalamic components involved in electrolyte and water homeostasis

Polyuria, hypernatremia, and hypovolemia are the major clinical signs of inherited nephrogenic diabetes insipidus (NDI). Hypernatremia is commonly considered a secondary sign caused by the net loss of water due to insufficient insertion of aquaporin-2 water channels into the apical membrane of the collecting duct cells. In the present study, we employed transcriptome-wide expression analysis to study gene expression in V2 vasopressin receptor (Avpr2)-deficient mice, an animal model for X-linked NDI. Gene expression changes in NDI mice indicate increased proximal tubular sodium reabsorption. Expression of several key genes including Na+-K+-ATPase and carbonic anhydrases was increased at the mRNA levels and accompanied by enhanced enzyme activities. In addition, altered expression was also observed for components of the eicosanoid and thyroid hormone pathways, including cyclooxygenases and deiodinases, in both kidney and hypothalamus. These effects are likely to contribute to the clinical NDI phenotype. Finally, our data highlight the involvement of the renin-angiotensin-aldosterone system in NDI pathophysiology and provide clues to explain the effectiveness of diuretics and indomethacin in the treatment of NDI.

A Novel Form of 6-Phosphofructokinase IDENTIFICATION AND FUNCTIONAL RELEVANCE OF A THIRD TYPE OF SUBUNIT IN PICHIA PASTORIS

Classically, 6-phosphofructokinases are homo- and hetero-oligomeric enzymes consisting of α subunits and α/β 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, α, β, and γ. The sequence of the γ subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the γ 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 γ subunit is not required for enzymatic activity, the γ 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 γ subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the γ subunit. Therefore, we suggest that the γ subunit contributes to adaptation of P. pastoris to energy resources.

6‐phosphofructokinase from Pichia pastoris: purification, kinetic and molecular characterization of the enzyme

6‐Phosphofructokinase from Pichia pastoris was purified for the first time to homogeneity applying seven steps, including pseudo‐affinity dye‐ligand chromatography on Procion Blue H‐5R‐Sepharose. The specific activity of the purified enzyme was about 80 U/mg. It behaves as a typically allosteric 6‐phosphofructokinase exhibiting activation by AMP and fructose 2,6‐bis(phosphate), inhibition by ATP and cooperativity to fructose 6‐phosphate. However, in comparison with the enzymes from Saccharomyces cerevisiae and Kluyveromyces lactis, the activation ratio of 6‐phosphofructokinase from Pichia pastoris by AMP is several times higher, the ATP inhibition is stronger and the apparent affinity to fructose 6‐phosphate is significantly lower. Aqueous two‐phase affinity partitioning with Cibacron Blue F3G‐A did not reflect remarkable structural differences of the nucleotide binding sites of the Pfks from Pichia pastoris and Saccharomyces cerevisiae. The structural organisation of the active enzyme seems to be different in comparison with hetero‐octameric 6‐phosphofructokinases from other yeast species. The enzyme was found to be a hetero‐oligomer with an molecular mass of 975 kDa (sedimentation equilibrium measurements) consisting of two distinct types of subunits in an equimolar ratio with molecular masses of 113 kDa and 98 kDa (SDS–PAGE), respectively, and a third non‐covalently complexed protein component (34 kDa, SDS–PAGE). The latter seems to be necessary for the catalytic activity of the enzyme. Sequencing of the N‐terminus (VTKDSIXRDLEXENXGXXFF) and of peptide fragments by applying MALDI–TOF PSD, m/z 1517.3 (DAMNVVNH) and m/z 2177.2 [AQNCNVC(L/I)SVHEAHTM] gave no relevant information about the identity of this protein. Copyright

Molecular genetics of 6-phosphofructokinase in Pichia pastoris

Previously, studies on glucose‐induced microautophagy in the methylotrophic yeast Pichia pastoris provided evidence that the glucose‐induced selective autophagy‐1‐protein is the α‐subunit of 6‐phosphofructokinase (Pfk), a key enzyme in the glycolytic pathway. In our work, we could clearly demonstrate that two types of subunits of Pfk exist in P. pastoris. Investigating the yeast cell‐free extract by Western blot analysis, two distinct signals of Pfk were obtained. In addition, we isolated a DNA sequence containing the complete ORF of PpPFK2 encoding the β‐subunit of Pfk from P. pastoris with a deduced molecular mass of 103.7 kDa. On the basis of these results, a hetero‐oligomeric structure of Pfk in P. pastoris became obvious. Because the molecular and kinetic properties of a homo‐oligomeric yeast Pfk appear to be more similar to those of mammalian Pfk, as described in the literature, our results are of interest for the growing number of studies on P. pastoris as a heterologous production system. Furthermore, the 3′‐ and 5′‐non‐coding regions of PpPFK2 were isolated and several putative binding sites for regulatory factors could be identified in the promoter region. The sequence has been deposited in the GenBank data library under Accession No. AF395078. Copyright

Structures of Schizosaccharomyces pombe Phosphofructokinase in the Presence of Either F6P or ATP

Phosphofructokinase (Pfk-1), the main regulatory enzyme of the glycolytic pathway, catalyzes the phosphorylation of fructose 6-phosphate (F6P) in the presence of magnesium and adenosine triphosphate (ATP). The key role that the enzyme plays in regulation is evidenced by the large number of allosteric effectors (activators and inhibitors) that control its activity in eukaryotic organisms. Eukaryotic phosphofructokinases differ in size and oligomerization state, and they also exhibit a concentration dependent association-dissociation behavior that is exploited as a regulatory mechanism.

Quantitative Characterization Of The Interactions Of Some Glycolytic Enzymes: An Application Of The Fluorescence Anisotropy Measurement

The affinity of Saccharomyces cerevisiae fructose-1,6-bisphosphate aldolase towards the metabolically related enzymes phosphofructokinase (PFK), triosephosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GPDH) was tested by using the signal of fluorescein isothiocyanate (FITC) attached covalently to the aldolase. The dissociation constants of the enzyme-enzyme complexes and rate constants of their formation and dissociation were measured and compared with the same parameters derived for enzymes from rabbit muscle. Hybrid complex formation between yeast aldolase and muscle GPDH and between muscle aldolase and yeast GPDH have also been observed. From the similarities in the determined parameters for the yeast and muscle enzymes we concluded that organization based on direct enzyme-enzyme interactions may be an ancient characteristic of the cytoplasm. The existence of in vitro hybrid complexes indicates that the recognition sites responsible for these interactions may have been conserved during the evolution.