Coding

Part:BBa_K5327008

Designed by: Fangxian Chen   Group: iGEM24_BUCT   (2024-08-26)


3-isopropylmalate dehydrogenase 1, chloroplastic

Function:[1][2][3]

Involved in both glucosinolate and leucine biosynthesis; catalyzes the oxidative decarboxylation step in both leucine biosynthesis (primary metabolism) and methionine chain elongation of glucosinolates (specialized metabolism).Catalyzes the oxidation of 3-carboxy-2-hydroxy-4-methylpentanoate (3-isopropylmalate, 3-IPM) to 3-carboxy-4-methyl-2-oxopentanoate. The product decarboxylates to 4-methyl-2 oxopentanoate.Required during pollen development and involved in embryo sac development. More active on 3-isopropylmalate and NAD+ than towards D-malate.

Usage and Biology

Genome localization:Chromosome 5 - NC_003076.8

Expression diagram:

Fig 1. The expression diagram of 3-isopropylmalate dehydrogenase 1, chloroplastic

Corresponding enzyme structure:

Fig 2. The corresponding enzyme structure of 3-isopropylmalate dehydrogenase 1, chloroplastic

The PCR result:

Fig 3. The PCR result of 3-isopropylmalate dehydrogenase 1, chloroplastic

Subcellular localization:[4]

Located in the plastid and chloroplast stroma of cells

Fig 4. The subcellular localization of 3-isopropylmalate dehydrogenase 1, chloroplastic

Dynamics data:

Table 1. The dynamics data of 3-isopropylmalate dehydrogenase 1, chloroplastic

Design Notes

The design of the IPMDH1 gene is based on the coding sequence (CDS) from Arabidopsis thaliana and has been codon-optimized for Saccharomyces cerevisiae (S288C) to ensure efficient expression in yeast. IPMDH1 plays a crucial role in both leucine and glucosinolate biosynthesis, catalyzing the oxidation of 3-isopropylmalate and contributing to leucine's primary metabolism and the methionine chain elongation steps in glucosinolate synthesis. Specifically, in this project, IPMDH1 catalyzes the conversion of alkylmalate into homoketo acid. IPMDH1 is essential for pollen and embryo sac development, exhibiting higher activity for 3-isopropylmalate and NAD+ than for D-malate.[5]To express this enzyme in yeast, the GPD promoter(BBa_K517001) and CYC1 terminato(BBa_K2368002) were selected to ensure high-level expression and mRNA stability. After design completion, the optimized gene was inserted into a vector and introduced into Saccharomyces cerevisiae S288C via homologous recombination, followed by screening and expression verification using a defective strain. This design aims to maximize the dehydrogenase activity of IPMDH1 in yeast, particularly in glucosinolate synthesis, thereby enhancing yeast's functionality as a metabolic engineering platform.

Plasmid

Fig 5. The plasmid expression of 3-isopropylmalate dehydrogenase 1, chloroplastic

Source

Arabidopsis thaliana


References

  1. ↑ SAWADA Y, KUWAHARA A, NAGANO M, et al. Omics-based approaches to methionine side chain elongation in Arabidopsis: characterization of the genes encoding methylthioalkylmalate isomerase and methylthioalkylmalate dehydrogenase [J]. Plant & cell physiology, 2009, 50(7): 1181-90.
  2. ↑ NOZAWA A, TAKANO J, MIWA K, et al. Cloning of cDNAs encoding isopropylmalate dehydrogenase from Arabidopsis thaliana and accumulation patterns of their transcripts [J]. Bioscience, biotechnology, and biochemistry, 2005, 69(4): 806-10.
  3. ↑ HE Y, CHEN L, ZHOU Y, et al. Functional characterization of Arabidopsis thaliana isopropylmalate dehydrogenases reveals their important roles in gametophyte development [J]. The New phytologist, 2011, 189(1): 160-75.
  4. ↑ HE Y, MAWHINNEY T P, PREUSS M L, et al. A redox-active isopropylmalate dehydrogenase functions in the biosynthesis of glucosinolates and leucine in Arabidopsis [J]. The Plant journal : for cell and molecular biology, 2009, 60(4): 679-90.
  5. ↑ HE Y, MAWHINNEY T P, PREUSS M L, et al. A redox-active isopropylmalate dehydrogenase functions in the biosynthesis of glucosinolates and leucine in Arabidopsis [J]. The Plant journal : for cell and molecular biology, 2009, 60(4): 679-90.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 423
    Illegal BglII site found at 617
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 390
    Illegal BsaI.rc site found at 447


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