Part:BBa_K1640018:Experience

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Part functionality

We have demonstrated the functionality of ChlM enzyme encoding magnesium protoporphyrin IX methyltransferase (lac+ChlM: BBa_K1640018), an enzyme within the third operon in the chlorophyll-a biosynthesis pathway. ChlM catalyses the methylation of a carboxyl group in magnesium-protoporphyrin IX using cofactor S-Adenosyl-L-methionine (SAM), yielding magnesium-protophorphyrin IX mono-methyl ester (3);

S-adenosyl-L-methionine + magnesium protoporphyrin IX ⇌ S-adenosyl-L-homocysteine + magnesium protoporphyrin IX 13-methyl ester.

Figure 5. Catalysis of Mg-protoporphyrin IX to Mg-protoporphyrin IX monomethylester by ChlM(4).

To demonstrate functionality of ChlM, we added a lac promoter creating a lac + ChlM composite part (Figure 4). This was transformed into E. coli strains DH5a, K12 and XL-blue. Protein was induced using auto-induction media ZYM5052 (5).

Functional assay of ChlM

Cell lysate containing over expressed ChlM was added with substrates and products were separated by FPLC (Figure 3).

Figure 6. FPLC chromatogram of the products of ChlM activity alongside positive and negative controls. The substrate MG-PPIX elutes at 3.04 mins, while the product Mg-PPIX mono-ester elutes at 4.22 mins.

The FPLC chromatogram data enabled determination of ChlM enzyme activity (Figure 3). This data enabled further determination of levels of Mg-Protoporphyrin IX converted to Mg-Protoporphyrin IX-ME by ChlM. In this instance, 26% of the precursor molecule Mg-Protoporphyrin IX had been catalysed by the enzyme ChlM into Mg-Protoporphyrin IX-ME.

When compared to ChlM pET (positive control) expression, which successfully converted 12% of the precursor molecule, it is apparent that our expression of ChlM was very successful.

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