Molecular genetics of 6‐phosphofructokinase in <i>Pichia pastoris</i>

Edelmann, Anke; Bär, Jörg

doi:10.1002/yea.889

Cited by 7 publications

(7 citation statements)

References 27 publications

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“…Li and was prepared according to standard protocols. [22] All chemicals were purchased from Sigma-Aldrich unless otherwise specified. The simple liposome was made from mono-unsaturated lipids (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC) purchased from Avanti Polar Lipids using protocols that have been described in the literature.…”

Section: Yeasts Liposomes and Reagentsmentioning

confidence: 99%

Real‐time molecular assessment on oxidative injury of single cells using Raman spectroscopy

Chang

Lin

Chen

et al. 2009

J Raman Spectroscopy

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Oxidative stress is encountered in many biological systems; the resultant oxidative injury plays a significant role in the pathogenesis of diverse diseases. Conventional measurements on oxidative injury are employed almost exclusively on a large population of cells either by counting the fraction of cell death or by observing the fluorometric change resulting from exogenous reagents, thereby lacking in molecular detail and temporal specificity. In this work we combine laser tweezers and Raman spectroscopy to observe the response of single cells to oxidative stress. By measuring the temporal changes of vibrational spectra of single optically trapped cells, we demonstrate a molecular-level assessment of cellular oxidative injury in real time, both qualitatively and quantitatively, without the introduction of exogenous reagents. The main experimental findings are supported by the observation of Raman spectra of intermediates and downstream products. The abrogation of the above changes by ascorbic acid further illustrates the therapeutic effect of antioxidants against cellular oxidative injury. This approach is extensible to studies exploring the biochemical transformation of single cells or intracellular organelles in response to various chemical or physical stimuli. With the aid of 'molecular fingerprints', single-cell Raman spectroscopy exhibits a great potential for accessing the chemical aspects of cellular bioactivity, yielding insight into pathophysiological processes and assisting the development of novel therapeutic interventions against diseases.

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Section: Yeasts Liposomes and Reagentsmentioning

confidence: 99%

Real‐time molecular assessment on oxidative injury of single cells using Raman spectroscopy

Chang

Lin

Chen

et al. 2009

J Raman Spectroscopy

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“…In addition, it possesses a unique structural organization. The enzyme is composed of three distinct subunits -- α, β and γ -- of 109 kDa, 104 kDa and 41 kDa, respectively, encoded by the PFK1 , PFK2 and PFK3 genes (Edelmann and Bar, 2002; Tanneberger, et al, 2007). The α-subunit (encoded by PFK1 ) was originally identified due to its involvement in the initial steps of peroxisomal microautophagy (Yuan, et al, 1997).…”

Section: Introductionmentioning

confidence: 99%

3D structure of phosphofructokinase from Pichia pastoris: Localization of the novel γ-subunits

Benjamin

Radermacher

Kirchberger

et al. 2009

Journal of Structural Biology

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The largest and one of the most complex ATP-dependent allosteric phosphofructokinase (Pfk) has been found in the methylotrophic yeast, Pichia pastoris. The enzyme is a hetero-oligomer (~ 1 MDa) composed of three distinct subunits (α, β and γ) with molecular masses of 109, 104 and 41 kDa, respectively. While the α- and β-subunits show sequence similarities to other phosphofructokinase subunits, the γ-subunit does not show high homology to any known protein in the databases. We have determined the first quaternary structure of P. pastoris phosphofructokinase by 3D electron microscopy. Random conical techniques and tomography have been instrumental to ascertain the quality of the sample preparations for structural studies and to obtain a reliable 3D structure. The final reconstruction of P. pastoris Pfk resembles its yeast counterparts with four additional densities, assigned to four γ-subunits, bridging the N-terminal domains of the four pairs of α- and β-subunits. Our data has evidenced novel interactions between the γ- and the α-subunits comparable in intensity to the interactions, shown by cross-linking and limited proteolytic degradation experiments, between the γ- and β-subunits. The structural data provides clear insights into the allosteric fine-tuned regulation of the enzyme by ATP and AMP observed in this yeast species.

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“…PCR was performed under the following conditions: Predenaturation at 95°C for 15 min was followed by cycles of denaturation at 94°C for 30 s, annealing beginning at 72°C for 30 s, and elongation at 72°C for 90 s. The annealing temperature was lowered 1°C per cycle to 50°C, which then was applied for annealing in the next 20 cycles. To identify the 5Ј-and 3Ј-ends, rapid amplification of cDNA ends (RACE)-PCR was performed as described previously (17) and according to the manufacturer's protocol (Gene-Racer TM kit with cloned avian myeloblastosis virus reverse transcriptase, Invitrogen). PCR fragments were subcloned into pCR2.1 (the TOPO T -…”

Section: Methodsmentioning

confidence: 99%

“…The ␤ subunit encoding gene, PpPFK2, was cloned recently (17), but sequence information of PpPFK1, encoding the PpPfk ␣ subunit, was lacking. We isolated a 6862-bp genomic fragment containing the complete coding sequence of PpPFK1 (2970 bp) and parts of the 5Ј and 3Ј non-coding regions (3809 and 83 bp, respectively) (GenBank TM accession number AF508861; supplemental Fig.…”

Section: Fru6-pmentioning

confidence: 99%

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A Novel Form of 6-Phosphofructokinase

Tanneberger

Kirchberger

Bär

et al. 2007

Journal of Biological Chemistry

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Classically, 6-phosphofructokinases are homo-and heterooligomeric 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 wildtype 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.The ATP-dependent 6-phosphofructokinase (EC 2.7.1.11, phosphofructokinase-1, ATP:D-fructose-6-phosphate 1-phosphotransferase (Pfk)) 2 catalyzes in many organisms the phosphorylation of fructose 6-phosphate (Fru 6-P) at position 1. The Pfk activity is generally being sensitive to a number of allosteric regulators, e.g. ATP, AMP, NH 4 ϩ , and fructose 2,6-bisphosphate (Fru 2,6-P 2 ). Therefore, this irreversible reaction is considered to be one of the rate-limiting steps of glycolysis (1-3). Most eukaryotic Pfks are heteromeric enzymes consisting of subunits, which evolved from a single ancestor gene by gene duplication and mutational events (4, 5). Specific amino acid residues involved in catalytic and regulatory functions of Pfk from Escherichia coli (6, 7) are conserved in yeast and mammalian Pfk genes. In eukaryotes the N-terminal half of a Pfk subunit obviously retained the catalytic function, whereas in the C-terminal half allosteric ligand binding sites have evolved from former catalytic and regulatory sites (4,8,9). This assumption is supported by studies with mutants of Saccharomyces cerevisiae expressing only the ␣ or the ␤ subunit of Pfk. It was demonstrated that one subunit type alone is able to form an enzymatically active Pfk entity in vivo (10, 11). Crystallographic analysis showed that an active bacterial Pfk consists of four identical subunits (12, 13). No high resolution structure of a eukaryotic Pfk is available yet. But electron microscopic studies with S. cerevisiae Pfk (ScPfk) at 10.8-Å resolution suggested an octameric enzyme assembly (14).Recently we co-purified a protein component together with the known Pfk ␣ and ␤ subunits (15-17) from the methylotrophic yeast Pichia pastoris. This unknown protein could only be separated under denaturating conditio...

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Molecular genetics of 6‐phosphofructokinase in Pichia pastoris

Cited by 7 publications

References 27 publications

Real‐time molecular assessment on oxidative injury of single cells using Raman spectroscopy

Real‐time molecular assessment on oxidative injury of single cells using Raman spectroscopy

3D structure of phosphofructokinase from Pichia pastoris: Localization of the novel γ-subunits

A Novel Form of 6-Phosphofructokinase

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