Mining of key genes involved in the sulforaphane biosynthetic pathway of moringa and cloning, expression, and functional verification of MoMYR1.
Level 5 - mechanism / opinion, no new human data
In vitro biochemical and plant molecular biology study without human subjects (CEBM level 5 by design analogy).
PubMed 41651260 · doi:10.1016/j.ijbiomac.2026.150633
What was done
Investigators mined the Moringa oleifera genome using local BLAST and KEGG pathway mapping to identify candidate genes involved in isothiocyanate biosynthesis. After tissue expression correlation analyses against sulforaphane content, the MoMYR1 gene was cloned and heterologously expressed in Escherichia coli BL21 (while MoMYR3 was tested in Pichia pastoris). The recombinant MoMYR1 protein was evaluated through molecular docking, in vitro kinetic/enzymatic assays with sinigrin and glucoraphanin substrates, and liquid chromatography-mass spectrometry (LC-MS).
What was found
Genomic analysis identified nine candidate genes across six enzyme classes (BCAT, CYP79F1, CYP83A1, UGT, SOT, and MYR). Only myrosinase genes MoMYR1 and MoMYR3 significantly positively correlated with tissue sulforaphane content (p < 0.05). Heterologous expression in E. coli produced recombinant MoMYR1 protein, whereas MoMYR3 yielded no detectable protein in P. pastoris. Molecular docking showed a substrate binding energy of -8.0 kcal/mol. In vitro assays demonstrated specific activities of 0.2565 and 0.2447 μmol·(min·mg)⁻¹ and catalytic efficiencies of 30.17 and 28.79 U·g⁻¹ for sinigrin and glucoraphanin, respectively. LC-MS confirmed recombinant MoMYR1 catalyzed the conversion of glucoraphanin to sulforaphane.
Why it matters
This study provides molecular and enzymatic characterization of MoMYR1 as a functional myrosinase in Moringa oleifera, establishing a key genetic target for understanding sulforaphane biosynthesis and developing bioproduction methods.
Limits
This was an exclusively benchtop in vitro and in silico study without in vivo plant knockout models or any animal/human testing. MoMYR3 could not be functionally characterized due to expression failure in yeast, and precise replicate counts and variance metrics were not detailed in the abstract.
Cited by
- supports Moringa, daikon radish, mustard seed, wasabi, and horseradish are rich sources of the enzyme myrosinase.