Study On Molecular Dynamics Simulations Of Key Enzyme PhzF And PhzG In Biosynthetic Pathway And Qsar Of Phenazine Antibiotics

Phenazines are a large class of well-known natural and synthetic nitrogen-containing heterocyclic compounds.In the past century,more than 6000 compounds that contain the phenazine ring system have been identified,of which over 100 are natural derivatives isolated as secondary metabolites,mainly from microorganisms such as Pseudomonas spp.and Streptomyces spp..Most phenazines show broad-spectrum bioactivities such as antibiotic activities against bacteria,fungi,yeast,and parasites,and antitumor activities.Studies of the effects of phenazines have shown that these compounds have multiple functions,and not merely bioactivities.Our lab have been studying the biosynthesis and regulation of phenazine compounds for many years.The shikimate pathway is believed to be primary pathway of phenazines biosynthesis.The synthesis involves chorismic acid conversion to phenazine-1,6-dicarboxylic acid and phenazine-1-carboxylic acid via 2-amino-2-desoxyisochorismic acid and trans-2,3-dihydro-3-hydroxyanthranilic acid(DHHA).Phenazine-1,6-dicarboxylic acid and phenazine-1-carboxylic acid are the precursors for more complex phenazine derivatives.Although the Phz F and Phz G crystal structure has been determined recently,an understanding of the detailed catalytic mechanism and the roles of key catalytic residues are still lacking.In study,we performed detailed computational investigations,using molecular dynamics and quantum mechanics/molecular mechanics methods,to simulate the interactions between atoms,to increase our understanding of the specific mechanism of Phz F and Phz G.The simulation results provide detailed information on the time-dependent conformational changes of proteins and the reaction process.This study provides new insights into the detailed catalytic mechanism of Phz F and Phz G.The following are main results:(1)Study on Structure and Function of Phz FIn this study,we performed a detailed computational investigation of Phz F using MD simulations,to understand the enzymatic isomerization reaction mechanism.The first two eigenvalues of the principal components for the Apo(Apo-enzyme complex),ES(Enzyme-substrate complex),ET(Enzyme-transition state analogous compound complex),and EP(Enzymeproduct complex)accounted for approximately 84%,78%,80%,and 79%of the total motions.During the simulations,RMSFs show that the backbone atoms of Apo,ES,ET,and EP were observed to have flexibilities similar to that of the initial crystal structure.The simulations showed high fluctuations of the structures of ?16 and L1,which form a passage leading to the active site,which was not observable from static crystal structures.Ser44 and Asp208 are key residues acting as door.(2)Study on Catalysis Process on Phz FIn a previously proposed mechanism,it was suggested that the Glu45 residue abstract the proton at C3 of the substrate to initiate the isomerization reaction.We built the reactant,Transition state 1(TS 1),intermediate,Transition state 2(TS 2)and product models to confirm this process.Our results proved that Glu45,acting as a general acid/base,is involved in proton transfer from C3 to C1.Our models revealed double roles of Glu45 in the isomerization reaction: it not only formed a hydrogen bond with the amino group at C2 of the substrate to help the enzyme bind and recognize the substrate,but also initiated the isomerization reaction by behaving as a proton shuttle.In addition,our models account for the important hydrogen-bonding network.We found that Asp208 did not participate directly in proton transfer but was hydrogen bonded with the amino group at C2 of the substrate.A water molecule was hydrogen bonded with the hydroxyl group at C3 of the substrate,and the residues Gly73,His74,Gly212,and Ser213 formed hydrogen bonds with carboxylic group oxygens,which helped to bind and stabilize the structure at the active site.(3)Study on MD Simulations of Phz GWe carried out Sitemap to predict active pocket of Phz G and used MD simulations to find enzymatic structural features.The hydrogen statistics showed reduced FMN(FMNR)have more hydrogen than oxidized FMN(FMNO).The detailed analysis indicated that FMNR form hydrogen bonds with Gln144 A and hydrogen occupancy accounted for 44.37%.But this didn't find in FMNO.During the simulations,both FMNO and FMNR can interact with Gln109 and Arg197 respectively by hydrogen bonds,which mean that Phz G function as dimer.In addition,we utilize quantitative structure-activity relationships(QSAR),docking models and MD simulations to predict how phenazine compounds bind to the target enzyme topoisomerase I,and to determine which functional groups are necessary for inhibiting the enzymatic activity.We build successfully 3D-QSAR and pharmacophore models,which mathematically and statistically correlates biological activities of phenazines with their chemical structure,and also help theoretically predict the activity of novel phenazine derivatives prior to testing and synthesis.The pharmacophore will be designed to find the common chemical features such as hydrogen bond acceptor,hydrogen bond donor,hydrophobic,aromatic ring,positive and negative charge shared by a series of phenazine compounds.We find Benophenazines have three function groups interact with DNA:(1)Aromatic ring to insert into DNA base plane;(2)Form hydrogen bond with Top I residues via substitution groups of 4-,8-,9-and10 position to anchor with DNA;(3)Tail of side chain with positive charge in major groove can interact with phosphate on DNA with negative charge by electrostatic interaction.Also,?-? conjugation make Benophenazine insert into base pairs more easily.