Part:BBa_K3875000

gadB-E89Q+△452-466

Usage and Biology

E.coli exploit the H+-consuming reaction catalysed by glutamate decarboxylase (Gad), which can converse glumate to GABA, to survive in acidic environment. This mechanism has been found in Escherichia coli for many years. There are many E.coli glutamate decarboxylase isoforms, and GadB is one of them. Structural studies on GadB have demonstrated that GadB undergoes pH-dependent changes in conformation, cellular localization and enzymatic activity, which combine to regulate the intracellular pH. And H+-dependent activation of GadB is influenced by N-terminal helix formation[1].
As we can see from the flow chart, Glucose and Acetyl-CoA can be consumed in the citric acid cycle to form ɑ-ketoglutaricacid, which can produce L-glutamate under the catalyst of gdhA, L-glutamate can be future turned into GABA(4-aminobutyricacid) under the catalyst of L-glutamate decarboxylase (GadB) [2].
As we said before, H+-dependent activation of GadB is influenced by N-terminal helix formation. Shukuya and Schwert has revealed that at pH 4.6, disordered C-termini and shifted β-hairpins allow full access of the substrate to the six active sites, while the N-termini are ordered and form two triple α-helical bundles[3].
It is these bundles are essential to recruit the protein under acidic conditions. On the other hand, at pH 7.6, the N-termini of all six subunits are disordered, which results in inactivation of GadB [1].
But GadB will remain active if it is mutant. Concerning that the wild-type GadB has an optimal activity at pH 4.5, which is not a suitable condition for cell growth. By mutating gadB to mutant gene (gadB E89Q Δ452-466), the GadB transcribed by our gene gadB(mut) is active in a wide range of pH, so as to overproduce GABA [4].

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 700
  
• 1000
  COMPATIBLE WITH RFC[1000]

References:

[1] Gut, H., Pennacchietti, E., John, R. A., Bossa, F., Capitani, G., De Biase, D., & Grütter, M. G. (2006). Escherichia coli acid resistance: pH-sensing, activation by chloride and autoinhibition in GadB. The EMBO journal, 25(11), 2643–2651. https://doi.org/10.1038/sj.emboj.7601107

[2] Chae, T. U., Ko, Y. S., Hwang, K. S., & Lee, S. Y. (2017). Metabolic engineering of Escherichia coli for the production of four-, five- and six-carbon lactams. Metabolic engineering, 41, 82–91. https://doi.org/10.1016/j.ymben.2017.04.001

[3] SHUKUYA, R., & SCHWERT, G. W. (1960). Glutamic acid decarboxylase. I. Isolation procedures and properties of the enzyme. The Journal of biological chemistry, 235, 1649–1652.

[4] Sheng, L., Shen, D., Yang, W., Zhang, M., Zeng, Y., Xu, J., Deng, X., & Cheng, Y. (2017). GABA Pathway Rate-Limit Citrate Degradation in Postharvest Citrus Fruit Evidence from HB Pumelo (Citrus grandis) × Fairchild (Citrus reticulata) Hybrid Population. Journal of agricultural and food chemistry, 65(8), 1669–1676. https://doi.org/10.1021/acs.jafc.6b05237