Part:BBa_K5406001
Polyphosphatate kinase (PPK)
Basic Information: This part contains the genetic sequence of Poly-Phosphatate Kinase (PPK henceforth) of the bacteria Thermosynechococcus vestitus (nee. elongatus) BP-1 or PPKTe. This sequence has been adapted to favor Escherichia coli (E.coli) expression and to also comply with the Type IIS Assembly Standard.
General Information: Core to the project of iGEM Athens 2024 is GshFss (see part BBa_K5406000), which uses ATP to produce Glutathione from its' composite enzymes. In our project, we want to create a system that produces ATP automonously. For that purpose we turned to PPKTe.
PPKTe is poly-phosphatase kinase. It is capable of producing ATP from ADP and polyphosphate (PolyP). PolyP is a bio-polymer which is compused from phosphate monomers linked by covalent bonds. Therefore, PPKTe catalyzes the reaction:
ADP + (PolyP)n --> ATP + (PolyP)n-1
Theoretical Biochemical Protein Parameters Information: Prior to the structural study of the enzyme PPKTe, we also made use of an online bioinformatics tool for the theoretical determination of important protein parameters, in order to have a clear view of PPKTe's key biochemical characteristics, that could be helpful for our experimental data analysis, as well. The ProtParam online tool of Expasy / the Swiss Institute for Bioinformatics – SIB was used for theoretical parameters calculation regarding protein biochemistry based on the amino acid sequence of a protein.
We used the amino acid sequence of PPK enzyme from Thermosynechococcus vestitus (nee. elongatus) BP-1 or PPKTe from the Protein database of the National Center for Biotechnology Information:
>BAC07766.1 polyphosphate kinase [Thermosynechococcus vestitus BP-1]
MEASVTNKQEQTAMPSAKSPRRKAPEPIDLDNPQYYFNRSLSWLEFNKRVLHEAYDPRTPLLERLKFMAIFSSNLDEFFMVRVAGLKQQVESGILQVGADGMP PAEQLQAVRQYLLPIVTEQHRYFDQELRALLAKESIFLTRFNELTPEQQAYLNDYFQAQVFPVLTPLAVDPAHPFPYISSLSLNLAVLIRDPESGQERLARVK VPNQFPRFVALPQHLHSPQGVHWLGVPLEEIIAHNLSALFPGMEIEAYFAFRITRSADLELETDKADDLLIAIEQEIRKRRFGSVVRLEVQRGIPPLLRQTLM EEMDLEEIDVYELEGLLCLNDLFAFMGLPLPQFKDPEWQPQVPPSFQRVEERESMFDTSSEITTLGTDYWEAVANELFSLIREGDIIVHHPYHSFAATVQRFI TLAAHDPQVLAIKITLYRTSGDSPIVSALIKAAENGKQVAVLVELKARFDEENNILWARKLEKVGVHVVYGVPGLKTHTKTVLVVRQEAGQIRRYVHIGTGNY NPKTASLYEDLGLFSCREELGADLSELFNVLTGYARQRDYRKLLVAPVTMRDRTLQLIYREIEHARNGQPARIIAKMNAITDTQVIRALYEASQAGVDIDLII RGMCCLRPGVPGVSDRIRVISIIGRFLEHSRIFYFGNNGDPEYYIGSADWRSRNLDRRVEAITPIEDPAIQLELKERLEIMLADNRQAWELQPDGTYRQRQPA PGEAERGTHSVLMARTLKDVQGSH
Using the provided sequence, the online algorithm of ProtParam calculated a set of basic specificities of the enzymatic biochemistry parameters, which can be seen in the tables below. More importantly, the total number as well as composition of amino acids contained in PPKTe are presented, along with the calculated Molecular Weight and Theoretical PI of the protein. Furthermore, indexes regarding the stability of the enzyme were also determined based on the amino acid sequence used as input. Such measures could be useful not only to have a broader knowledge of a biomolecule, but also to tailor further experiments in the future, for example conducting targeted mutagenesis to meet specific requirements, for example enhanced enzyme activity or even thermostability.
Amino Acid Number: 745 Theoretical PI: 5.64 Molecular Weight 84986.26 Da or 85 kDa Estimated Half-Life: >10 h (E. coli in vivo) or > 20 h (Yeast, in vivo) or 30 h (mammalian reticulocytes, in vitro) Instability Index: 44.81 - Instable Aliphatic Index: 96.48 GRAVY (Grand average of hydropathicity): -0.263
Amino Acid Composition:
Amino Acid/ Number of Residues/ Percentage % in Sequence
Alanine / 60 / 8.1%
Arginine / 59 / 7.9%
Asparagine / 24 / 3.2%
Aspartic acid / 38 / 5.1%
Cysteine / 4 / 0.5%
Glutamine / 42 / 5.6%
Glutamic acid / 62 / 8.3%
Glycine / 38 / 5.1%
Histidine / 18 / 2.4%
Isoleucine / 47 / 6.3%
Leucine / 84 / 11.3%
Lysine / 24 / 3.2%
Methionine / 15 / 2.0%
Phenylalanine / 32 / 4.3%
Proline / 46 / 6.2%
Serine / 37 / 5.0%
Threonine / 32 / 4.3%
Tryptophan / 7 / 0.9%
Tyrosine / 25 / 3.4%
Valine / 51 / 6.8%
Total of negatively charged amino acids: (Asp + Glu) 100
Total of positively charged amino acids: (Arg + Lys) 83
Structural Information:
In order to obtain a representative model protein structure of PPKTe from Thermosynechococcus vestitus (nee. elongatus) BP-1, we employed online bioinformatic tools for homology modeling. More specifically, we used the SWISS-MODEL protein structure homology-modeling server of Expasy / the Swiss Institute for Bioinformatics – SIB. We used the amino-acid sequence of PPKTe as an input, leading to the export of a model protein structure based on homology.
After the alignment of a model structure of the input PPKTe with templates from the server, the results of the homology modeling analysis through SWISS-MODEL showed that in fact the most suitable model based on homology for PPK was in fact the protein structure of Polyphosphate kinase from Escherichia coli, enlisted as 1XDO in the Protein Data Bank. This structure was chosen as a template for the alignment with the PPKTe model, showing that both structures are homo-dimers with a sequence identity of 33.68%. The structures taken for both the model homo-dimer structure of PPKTe as well as the template from Escherichia coli are shown in the respective pictures below.
To further support our homology modeling results from both blastp amino-acid sequence alignments as well as SWISS-MODEL, we also harnessed the AlphaFold Protein Structure Database, which is a novel online AI tool (Google DeepMind - EMBL-EBI) that predicts a protein’s 3D structure from its amino acid sequence. We used the UniProt accession number of PPK from Thermosynechococcus vestitus (nee. elongatus) BP-1 - Q8DMA8 to get a structure assessment based on AI from AlphaFold, depicting PPKTe in the form of a monomer.
Finally, for our first effort in achieving a 3D visualization for the enzyme PPKTe from the microorganism Thermosynechococcus vestitus (nee. elongatus) BP-1, we used the PyMol (™) © Schrödinger molecular visualization system. For this purpose, we used the .pdb file structures obtained from AlphaFold, and thus the 3D structure of PPKTe as a monomer, based on homology modeling is presented in the following picture.
To make the comparison between homologous and model structures more clear, we are presenting the model of the PPKTe monomer (colored in lime green) in alignment with the homologous protein structure of the homodimer enzyme PPK from Escherichia coli, listed as 1XDO in PDB (colored in light pink), using the align tool of PyMol.
Any use of Pymol Software was strictly done for educational and scientific purposes under Fair Use Provisions of US and EU Law.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 311
Illegal PstI site found at 1708 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 311
Illegal PstI site found at 1708 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2052
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 311
Illegal PstI site found at 1708 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 311
Illegal PstI site found at 1708 - 1000COMPATIBLE WITH RFC[1000]
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