Caffeic acid O-methyltransferase from Leucaena leucocephala: Cloning, expression, characterization and molecular docking analyses

• cDNA of OMT (1098 bp) from Leucaena leucocephala stem was cloned.
• Recombinant LlCOMT was expressed, purified and characterized.
• Based on molecular docking, thiol esters were found to be best substrates.

Angiospermic caffeic acid O-methyltransferase (COMT) conventionally catalyzes the methylation of caffeic acid as well as 5-hydroxy ferulic acid to ferulic acid and sinapic acid, respectively, using S-adenosyl-l-methionine (SAM) as methyl group donor. A cDNA of OMT from Leucaena leucocephala (LlOMT) was cloned, expressed and purified. The expressed protein was purified to homogeneity on Ni–NTA agarose column with specific activity of 450 nmol of ferulic acid formed/min/mg protein. Native molecular weight of the purified enzyme was found to be 80 kDa and that of subunit molecular weight was found to be 40 kDa, suggesting the homodimeric nature of the enzyme. The optimum temperature and pH was found to be 37 °C and pH 8.0, respectively. Apparent Km of enzyme for caffeic acid and SAM was found to be 220 μM and 2.12 μM, respectively. In view of overlapping/metabolic grid concept of methylation of substrates in phenyl propanoid pathway, an in silico approach was used to look into it. Accordingly, the LlOMT protein was modeled and docked with 16 putative substrates (intermediates of phenyl propanoid/monolignol biosynthesis pathway). In silico analyses revealed that the Co-A ester substrates were most favored among those of substrates, analyzed.

3D structure of modeled LlCOMT docked with methyl donor (SAM) and best putative substrate (5-hydroxy feruloyl CoA) as ligands.Figure optionsDownload as PowerPoint slide