EG10699

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Author Information

Selin Chiragzada

Basic Information

  • ID: EG10699
  • Name: pfkA (ATP-dependent 6-phosphofructokinase)
  • Organism: E. coli
  • Description:

The gene produces the ATP-dependent 6-phosphofructokinase enzyme. 6-phosphofructokinase catalyzes the first step of glycolysis, that is, the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate by ATP:

ATP + D-fructose 6-phosphate = ADP + D-fructose 1,6-bisphosphate

The enzyme uses Mg2+ as a cofactor (http://www.uniprot.org/uniprot/F4MPI1). Its activity is allosterically regulated: ATP and phosphoenolpyruvate inhibit it and ADP activates it, depending on the cell’s needs. 6-phosphofructokinase has shown to have 2 lobes, alpha and beta: one binds ATP and the other is involved in substrate-bindings and contains the allosteric site. It exists as a homotetramer in mammals and bacteria, meaning that each monomer contains two identical domains. The domains have 2 main states - the ‘closed’ state and the ‘open state’. In the ‘closed’ state, the magnesium ion brings the two products of the reaction closer together. In the ‘open state’, only ADP binds to the magnesium, which drives the products further apart from each other. These stages are thought to be the successive stages of the reaction (https://en.wikipedia.org/wiki/Phosphofructokinase).

  • DNA Length: 963 base pairs.
  • DNA sequence:

ATG ATA AAG AAA ATC GGG GTT CTT ACT TCA GGA GGC GAT GCC CCA GGT ATG AAT GCG GCT ATT CGT GGT GTG GTT CGG TCG GCT TTG ACT GAA GGA TTA GAA GTG ATG GGC ATC TAT GAC GGC TAC CTT GGC TTG TAT GAA GAT CGG ATG GTT CAG CTG GAC CGG TAT TCC GTC TCC GAT ATG ATC AAC AGA GGT GGG ACC TTT TTG GGC AGT GCC AGA TTC CCT GAG TTT CGG GAT GAG AAC ATC CGT GCC GTG GCC ATC GAG AAC CTG AAG AAG CGG GGG ATA GAC GCT TTG GTG GTT ATT GGG GGC GAT GGT TCC TAT ATG GGT GCT ATG AGA CTG ACG GAG ATG GGT TTT CCA TGC ATT GGA TTA CCA GGA ACG ATA GAC AAT GAT ATA AAG GGC ACA GAC TAC ACC ATA GGC TTC TTT ACC GCC TTG TCT ACT GTC GTA GAA GCA ATA GAT CGC CTT AGA GAC ACT TCC AGT TCT CAT CAG CGT ATA AGC GTG GTC GAG GTT ATG GGC CGT TAT TGC GGA GAT CTG ACT TTA GCA GCA GCT ATC GCT GGA GGC TGT GAG TTC GTT GTA GTG CCT GAA GTC GAG TTT AGT CGG GAG GAC CTG GTC AAT GAG ATC AAA GCC GGT ATA GCC AAA GGC AAG AAA CAT GCG ATT GTT GCT ATT ACG GAA CAC ATG TGC GAC GTT GAT GAG CTG GCC CAC TTT ATT GAA AAA GAA ACC GGT CGT GAG ACT AGA GCG ACG GTA TTG GGG CAT ATT CAA CGC GGG GGG AGC CCC GTA CCT TAC GAC CGT ATA TTA GCG AGC CGT ATG GGC GCC TAC GCG ATT GAT CTT CTG TTA GCC GGT TAT GGG GGG CGC TGT GTG GGC ATA CAG AAT GAA CAG TTA GTC CAT CAT GAC ATT ATC GAT GCG ATA GAG AAT ATG AAA CGG CCA TTT AAA GGG GAC TGG CTT GAT TGC GCG AAG AAG TTG TAT TAG

  • Amino Acid length: 320 amino acids.
  • Amino Acid sequence:

MIKKIGVLTS GGDAPGMNAA IRGVVRSALT EGLEVMGIYD GYLGLYEDRM VQLDRYSVSD MINRGGTFLG SARFPEFRDE NIRAVAIENL KKRGIDALVV IGGDGSYMGA MRLTEMGFPC IGLPGTIDND IKGTDYTIGF FTALSTVVEA IDRLRDTSSS HQRISVVEVM GRYCGDLTLA AAIAGGCEFV VVPEVEFSRE DLVNEIKAGI AKGKKHAIVA ITEHMCDVDE LAHFIEKETG RETRATVLGH IQRGGSPVPY DRILASRMGA YAIDLLLAGY GGRCVGIQNE QLVHHDIIDA IENMKRPFKG DWLDCAKKLY

Function and Homologs

  • Product: ATP-dependent 6-phosphofructokinase isozyme 1
  • Module: glycolitic pathway
  • Closest homologous proteins: The top (max three) homologous proteins to this protein, as identified by BLAST searches.
    • 6-phosphofructokinase [Escherichia coli MS 84-1], Max score = 651/Query Cover = 100%/E-Value = 0.00 /Ident = 100%, [EFJ85506.1]
    • 6-phosphofructokinase [Escherichia coli KO11FL], Max score = 651/Query Cover = 100%/E-Value = 0.00 /Ident = 100%, [ADX52955.1]
    • 6-phosphofructokinase I [Shigella flexneri 5 str. 8401], Max score = 651/Query Cover = 100%/E-Value = 0.00 /Ident = 100%, [ABF05609.1]
  • Equivalent E. coli / JCVI functional protein: MMSYN1_0220

Expression

  • Expression Level: high
  • Expression Level Hypothesis: The gene is highly expressed because the enzyme that it codes for plays an important role in glycolysis, a vital process for any cell. A possible explanation as to why this gene is highly expressed while its JCVI homolog has a low expression level is that E.coli primarily uses 6-phosphofructokinase as its main glycolysis enzyme, and JCVI may use a combination of different ones, meaning that it needs little of it.
  • Expression Level References and Description: I used the E.coli proteome dataset (File:EcoliProteomicExpressionData.xlsx) and I'm sure about my choice since I was able to find the exact gene in the given database.
  • Expression Time: early
  • Expression Level Hypothesis: 6-phosphofructokinase plays an important role in respiration, which is a vital process that occurs in all cells constantly. However, it would be more important for pfkA to be expressed early on in the development of the organism, so that it has a chance to grow.
  • Expression Time References and Description: I made this decision based on the protein's function & did not use any resources.

Gene Context

  • Other Components: EG12296
  • Possible Dependencies: enzymes, such as amylase, maltase and sucrase, that break down polysaccharide chains into glucose. Glucose is required for glycolysis to be able to occur.
  • Process: glycolysis (the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate)

Construct

  • Synthesis Score: The synthesis score of your construct: 1, 2,3
  • Predicted Translation Rate: Prediction of construct translation rate from the RBS calculator
  • Design Notes and Details: For example, had to use a rare codon to fix folding energy;
  • GenBank File: A link to the GenBank file. file