Coding

Part:BBa_K5327004

Designed by: Han Cao   Group: iGEM24_BUCT   (2024-08-20)


3-isopropylmalate dehydratase small subunit 2

Function:[1][2]

Catalyzes the isomerization between 2-isopropylmalate and 3-isopropylmalate, via the formation of 2-isopropylmaleate (Probable). Functions redundantly with LEUD2 in the methionine chain elongation pathway of aliphatic glucosinolate formation.

Usage and Biology

Genome localization: Chromosome 2 - NC_003071.7

Expression diagram:

Fig 1. The expression diagram of 3-isopropylmalate dehydratase small subunit 2

Corresponding enzyme structure:

Fig 2. The corresponding enzyme structure of 3-isopropylmalate dehydratase small subunit 2

The PCR result:

Fig 3. The PCR result of 3-isopropylmalate dehydratase small subunit 2

Subcellular localization:[3]

Located in the plastid and chloroplast stroma of cells

Fig 4. The subcellular localization of 3-isopropylmalate dehydratase small subunit 2

Design Notes

IPMI is a heterodimer composed of a large subunit (LSU) and a small subunit (SSU) that catalyzes the isomerization between 2-isopropylmalate and 3-isopropylmalate. In Arabidopsis thaliana, there is one LSU gene and three SSU genes,[4]with LSU playing an important role in methionine chain elongation and leucine biosynthesis. SSU2 and SSU3 have the ability to catalyze methionine chain elongation, while SSU1 is a crucial component of leucine biosynthesis. [5] We selected LSU1 and SSU2 to construct our pathway. To ensure efficient expression of 3-isopropylmalate dehydratase small subunit 2（SSU2） in Saccharomyces cerevisiae (S288C), we codon-optimized the SSU2 gene from Arabidopsis thaliana. SSU2 catalyzes the isomerization between 2-isopropylmalate and 3-isopropylmalate, possibly through the formation of 2-isopropylmaleate, and functions redundantly with LEUD2 in the methionine chain elongation pathway of aliphatic glucosinolate formation. We selected the ADH1 promoter (ADH1pBBa_J63005) and PYK1 terminator (PYK1tBBa_K5327018) to drive and stabilize gene expression. After designing the optimized gene, it was inserted into a vector and integrated into yeast via homologous recombination, followed by screening for defects and expression verification. This design strategy ensures that SSU2 efficiently catalyzes key isomerization reactions in the methionine chain elongation pathway, thereby promoting the efficient synthesis of aliphatic glucosinolates.

Plasmid

Fig 5. The plasmid expression of 3-isopropylmalate dehydratase small subunit 2

Source

Arabidopsis thaliana


References

  1. ↑ KNILL T, REICHELT M, PAETZ C, et al. Arabidopsis thaliana encodes a bacterial-type heterodimeric isopropylmalate isomerase involved in both Leu biosynthesis and the Met chain elongation pathway of glucosinolate formation [J]. Plant molecular biology, 2009, 71(3): 227-39.
  2. ↑ LäCHLER K, CLAUSS K, IMHOF J, et al. In Arabidopsis thaliana Substrate Recognition and Tissue- as Well as Plastid Type-Specific Expression Define the Roles of Distinct Small Subunits of Isopropylmalate Isomerase [J]. Frontiers in plant science, 2020, 11: 808.
  3. ↑ https://www.uniprot.org/uniprotkb/Q9ZW84/entry
  4. ↑ HE Y, CHEN B, PANG Q, et al. Functional specification of Arabidopsis isopropylmalate isomerases in glucosinolate and leucine biosynthesis [J]. Plant & cell physiology, 2010, 51(9): 1480-7.
  5. ↑ KNILL T, REICHELT M, PAETZ C, et al. Arabidopsis thaliana encodes a bacterial-type heterodimeric isopropylmalate isomerase involved in both Leu biosynthesis and the Met chain elongation pathway of glucosinolate formation [J]. Plant molecular biology, 2009, 71(3): 227-39.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 565
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 661
    Illegal AgeI site found at 702
  • 1000
    COMPATIBLE WITH RFC[1000]


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