Exploration Of Molecular Dynamics In Catalytic Promiscuity Of Important Glycoside Hydrolytic Enzymes

Enzymes,the driving force of life metabolism,catalyze various chemical reactions in living organisms.Industrial biotechnology with bio-catalysis as its core has become the third wave of modern biotechnology development.The efficiency of enzymes which can be improved by design and modification is the limiting bottleneck in industrial production.Important issues that influence the catalytic efficiency of enzymes is stability and substrate specificity.The non-specificity is also known as promiscuity,referring to the ability of an enzyme to catalyze many different substrates essentially.Glycoside hydrolases(GH)are widely used in industrial production,however,the specificity of GH is not strong,and they are highly promiscuous,which affects their catalytic efficiency or product yield.Therefore,it is necessary to reduce the enzyme promiscuity and improve the product specificity by rational design.In this study,the dynamic mechanism of enzymes in GH11,GH12,GH29 family was studied by using structural bioinformatics methods and molecular dynamics simulation.Besides,BfNCTC9343 in GH29 family,was modified by computer-aided screening combining with wet experiments,and its promiscuity was highly induced.The research work and main achievements of this paper are as follows;(1)Molecular dynamics simulation experiments were carried out on GH11 and GH12 families with similar topological structures and different functions.The results showed that the molecular dynamics of the active site architectures are an important factor causing the differences in the functional promiscuity of two families.The conformation distribution of amino acids in the active site architecture of GH12 family is diverse and dispersed,which is conducive to the binding of enzymes to different substrates.(2)The conformation of amino acids on the subsites of the active site architectures of GH11 and GH12 family enzymes was characterized and classified.The results showed that the active site architectures of enzymes in GH12 family have a large number of conformational diverse amino acid residues,and these residues are all over the active site architectures,which are conducive to the binding of enzymes to different substrates?(3)From the flexibility analysis of the TrCel12A and HsCel12A in GH12 family we found that Loop3 in TrCel12A has an obvious property of high flexibility,which is also found in other promiscuous enzymes in GH12 family.Binding energy calculation and molecular docking experiments further showed that the high flexibility of Loop3 can reduce the probability of forming steric hindrance between the substrate and TrCel12A,enabling the TrCel12A binding to the xyloglucan.(4)From the molecular dynamics simulations of BfNCTC9343 in the GH29 family we found that the temperature rise led to the change of the flexibility of Loop4 in BfNCTC9343,which affected the active site architecture molecular dynamics Then the distance between the key amino acid 229D and 288E at Loop4 increased.The probability of substrate conformation transition also increased,finally resulting in the promiscuity change of the BfNCTC9343.(5)We found a mutant through the computer-aided screen,L224H,which shows low flexibility in Loop4,which can reduce the promiscuity of BfNCTC9343.This also proves that the molecular dynamics of active site architecture and loops play a role in enzyme promiscuity.