Dataset Information

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

ABSTRACT: 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 ( approximately 1MDa) composed of three distinct subunits (alpha, beta and gamma) with molecular masses of 109, 104 and 41kDa, respectively. While the alpha- and beta-subunits show sequence similarities to other phosphofructokinase subunits, the gamma-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 gamma-subunits, bridging the N-terminal domains of the four pairs of alpha- and beta-subunits. Our data has evidenced novel interactions between the gamma- and the alpha-subunits comparable in intensity to the interactions, shown by cross-linking and limited proteolytic degradation experiments, between the gamma- and beta-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.

SUBMITTER: Benjamin S

PROVIDER: S-EPMC3586196 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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

Benjamin Shaun S Radermacher Michael M Kirchberger Jürgen J Schöneberg Torsten T Edelmann Anke A Ruiz Teresa T

Journal of structural biology 20090625 2

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 ( approximately 1MDa) composed of three distinct subunits (alpha, beta and gamma) with molecular masses of 109, 104 and 41kDa, respectively. While the alpha- and beta-subunits show sequence similarities to other phosphofructokinase subunits, the gamma-subunit does not show high homology to any known protein in the ...[more]

PMID: 19559794

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Project description:Over the past three decades, due to the universal application of Pichia yeast in the fermentation industry as well as the establishment of Pichia pastoris fermentation process for more than 30 years, the technology of the whole process has become very mature and has now reached a stagnated period of growth. However, studies and research conducted on the genomics of the classic fermentation process and the uncovering of the biological phenomena in the fermentation process from the point of view of high-throughput gene or protein is still in its early stages, and there is still insufficient data within this field. First, in collaboration with Agilent Company, we designed and prepared an expression microarray that could be used for the detection of Pichia pastoris transcriptomics. The transcriptomic changes in the five key technology steps (time points), during the fermentation process of Pichia pastoris would then be detected with the aid of an expression microarray. The five key steps of technology described above formed two important biological processes, namely, the limiting carbon source replacement and secondly, the fermentative production of exogenous proteins. The biological phenomena involved in these two processes were displayed and analyzed at the transcriptional level. In addition to this, with regard to the most important function in the fermentation process of Pichia pastoris, oxid-reduction, the metabolic drift process was analyzed and the important genes that might dominate the changes in the metabolic flux were discovered creatively by using the function tree method in this paper. This study was undertaken from the point of view of the transcriptome and the biological phenomena in the fermemntation process of Pichia pastoris. Both of which, were thoroughly explained during this study. The hope is for many more researchers to optimize the strain fermentation process, to produce proteins at the genetic level, as well as providing and obtaining new perspectives and detailed scientific data for the continued development within this field.

Dataset Information

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

Publications

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

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