The Molecular Mechanism Underlying The Synergistic Action Of A Multimodular Pectin Methylesterase/Polygalacturonase In The Pectin Degradation

Pectin,which is widely present in the cell walls of higher plants,is a kind of acidic complex polysaccharide.Pectinase,as a kind of mixed enzyme that specifically hydrolyzes pectin substrates,can completely hydrolyze pectin polysaccharides and is widely used in many industries such as fruit juice clarification.According to the different ways of acting on the substrate,pectinase can be divided into two categories: esterase and depolymerase.Of them,pectin methylesterase acts on the methyl esterified pectin substrate to catalyze the production of pectinic acid and methanol from methoxy esters.Polygalacturonase acts on the ?-1,4-D-glycosidic bond of the polygalacturonic acid chain to produce single or oligomeric galacturonic acid.The study of the molecular mechanism of the synergistic catalysis of the multifunctional pectinase that efficiently degrades pectin polysaccharides will not only deepen the regular understanding of the bifunctional pectin methylesterase/polygalacturonase synergistic degradation of pectin,but also provides a solid scientific basis for the accurate application of pectinase and the rational design of highefficiency bifunction pectinase.In this study,the bifunctional pectinase S6 A from the fungus Penicillium oxalicum SX6 with separate pectin methylesterase and polygalacturonase catalytic domains was used as the core material.Firstly,the catalytic mechanism of pectin methylesterase S6 PE in N-terminal domain was studied.Based on the reported crystal structure information,molecular replacement technology was used to obtain the model complex structure of S6 PE with the decasaccharide substrate.The residues related to the substrate binding in the entire catalytic channel were statistically analyzed and the functional verification of the key amino acids was carried out.The results showed that the sites in the catalytic channel(Q156,V199,F203,Q205,R261,W263,R264,W288 and D124)together maintained the correct active conformation of the substrate molecule.The substrate-specific catalytic mechanism of S6 PE has been determined: The sugar ring at sites-3 to-1 prefers galacturonic acid with no side chain modification,while the sugar ring at sites +1 to+3 prefers galacturonic acid with methyl ester modification on the side chain.Then,a similar strategy was used to study the catalytic mechanism of polygalacturonase S6 PG in the C-terminal domain of the bifunctional pectinase S6 A.The structure model of the complex of S6 PG with the tetrasaccharide substrate Gal A4 was obtained by molecular docking technology,and the functional verification analysis was performed on the amino acids related to substrate binding in S6 PG.The results showed that S6 PG has a strict preference for pectin substrates without any modification of side chain.The carboxyl group of the sugar ring Gal A located at the +1,-2 and-3 positions were fixed by R666,K668,H633,Y701,H551,S618 and S595,respectively.The enzyme activity was greatly reduced or lost after being mutated to alanine,suggested that the recognition of the carboxyl group of the substrate was mainly realized by the above amino acids.In addition,the hydroxyl group on the substrate also formed the hydrogen bond network with the key amino acids in S6 PG,maintaining the conformational stability of the sugar ring during the catalysis process.Finally,the molecular mechanism of the synergistic hydrolysis of pectin molecules by S6 PE and S6 PG in the bifunctional pectinase S6 A was studied.S6 PE was the rate-limiting enzyme of pectin degradation,the amount of reducing sugars produced by S6 PE and S6 PG was 5-fold higher than that of S6 PG released.In the bifunctional pectinase S6 A,S6PE and S6 PG hydrolyze the pectin substrate at the same time and greatly improved the hydrolysis efficiency by means of intramolecular synergistic interactions.After reaction 120 min,the total reducing sugar released by S6 A was 1.6 times higher than that of the 1:1 combination of S6 PE and S6 PG.Towards crude citrus peel and apple pomace pectin(5mg/m L)as substrates,the amount of reducing sugars produced by the bifunctional enzyme S6 A were7,400 and 7,600 ?M,respectively,and the final hydrolyzed products were monosaccharides,disaccharides,trisaccharides,and trisaccharide and tetrasaccharide with a methyl ester modification at the end of the sugar ring.It provided the theoretical basis for recycling of food processing by-products.