Characterization of charged functional domains introduced into a modified pectic homogalacturonan by an acidic plant pectin methylesterase (Ficus awkeotsang Makino) and modeling of enzyme mode of action

Abstract An acidic plant pectin methylesterase from Ficus awkeotsang achenes ( Fa PME) was used to demethylesterify a model homogalacturonan (HG) at pH 4.5 and 7.5. Introduced demethylesterified blocks (DMBs) were released by a limited endo-polygalacturonase (EPG) digestion, separated and quantified by HPAEC. The average DMB size ( B S ¯ ) and number of such blocks per molecule ( B N ¯ ) differed depending on the degree of methylesterification (DM) and reaction pH ( P B S ¯ and B N ¯ were observed in HGs of 30% DM compared to higher DMs. HGs demethylesterified to 30% and 50% DM at pH 4.5 showed significantly larger B S ¯ compared to pH 7.5. Absolute degree of blockiness (DB abs ), obtained using exhaustive EPG digestions, displayed a linear relationship with the DM regardless of reaction pH ( P in silico modeled enzyme mode of action suggested that a random, multiple chain, non-processive mode of action best explains the distributions of small blocks ( B S ¯ ≥ 11 ) and a processive multiple attack mechanism best explains the distributions of longer blocks. Decreasing the DM of the HGs by the Fa PME increased the G ′ and G ″ of calcium-mediated gels. Pearson's correlation displayed significant correlation coefficients between B S ¯ , B N ¯ , DB abs , DM, and G ′. The results suggest the possibility to control B S ¯ and to produce a uniform population of demethylesterified pectin molecules, particularly in acidic environments where most basic plant PMEs are less active.