Biochemical characterization of a key step involved in 2H4MB production in Decalepis hamiltonii

Decalepis hamiltonii is widely known for its flavour molecule 2-Hydroxy-4-Methoxy Benzaldehyde (2H4MB), a structural isomer of vanillin. As the biosynthetic pathway of 2H4MB is not known, we hypothesised 2H4MB origins could be from phenylpropanoid pathway (PPP). Accordingly, a study was conducted using PPP inhibitors (viz. piperonylic acid, MDCA and propanil) against in vitro root cultures of D. hamiltonii to find the branch of PPP which catalyses the 2H4MB formation. HPLC analysis was carried out to quantify 2H4MB levels in control and respective inhibitor treated root cultures in vitro. The results obtained revealed that piperonylic acid did not inhibit 2H4MB biosynthesis in the given period, whereas MDCA and propanil had the marked inhibitory effect. The inhibitory effect was evident with 13.2, 33.6 and 37.9% decrease in 2H4MB levels at 50, 100 and 150mM concentration of MDCA respectively in comparison with control roots. Similarly, the inhibitory effect of propanil on 2H4MB biosynthesis was obvious with 23.7, 49.5 and 57.9% decrease in 2H4MB levels at 50, 100 and 150μM concentration of inhibitor respectively when compared with control roots. Propanil showed a greater slow down effect on 2H4MB biosynthesis compared to MDCA. Incorporation of 0.1, 0.5 and 1.0mM ferulic acid as a precursor to in vitro root cultures of D. hamiltonii showed an increase in 2H4MB levels at the rate of 3.1, 107 and 94.1% respectively as quantified by HPLC analysis. However, ferulic acid in conjunction with propanil did not show any increase in 2H4MB levels. This clearly explains that ferulic acid is channelled through the 4-CL (4-coumarate CoA ligase) enzyme, where it would be converted to feruloyl-CoA and could be further converted to 2H4MB in D. hamiltonii.