Mechanisms Underlying Azoreduction By Shewanella Oneidensis MR-1 And Investigation Of Active Sites In Azoreductase

In China,textile industry and printing and dyeing industry are major users of dyes.Along with the increasing usage amount of dyes,the environmental pollution caused by wastewater discharge,which contains a mass of dyes,is becoming a severe issue.Azo dyes used by printing and dyeing industry are resistant to degrade under natural conditions.Azo dyes are toxic,mutagenic and carcinogenic and thus,have potential risks to living things.Shewanella oneidensis MR-1 are capable of degrading azo dyes under microaerobic conditions.However,the azoreducion process is largely inhibited under aerobic conditions.Neither the mechanisms underlying biodegradation of azo dyes by S.oneidensis MR-1 nor the mechanisms underlying inhibition by dissolved oxygen(DO)have been extensively explored.Azoreductase of S.oneidensis MR-1 is FMN dependent and show obvious preference for NADH.Nevertheless,the structure of azoreductase has not been successfully crystallized.Little information is known about the binding mode of ligands with azoreductase.In order to unravel these unkown mechanisms metioned above,metabonomic analysis based on NMR and trascriptome analysis based on RNA-Seq are employed to investigate the metabolic changes and transcriptional changes of S.oneidensis MR-1 cultured under different conditions,respectively.Homologous modeling by Modeller,molecular docking by AutoDock Vina and site-directed mutagenesis are adopted to investigate the active site of soAzoR and binding mode of FMN,NADH and methyl red(MR).All the results are demonstrated as below:1.Metabolome analysis revealed significant alteration(p<0.05)in the concentrations of eight metabolites,including lipoprotein lipids,lsoamyl acetate,lsopropanol,allothereonine,acetoin,alanine,putrescine and NAD+,when strain MR-1 was cultured under aerobic conditions.However,methyl orange exposure showed no obvious effect on metabolites of strain MR-1 under MAMO conditions.2.At the transcriptional level,genes involved in certain metabolic processes,particularly the tricarboxylic acid(TCA)cycle,amino acid biodegradation,urea cycle,biosynthesis of arginine and the electron transfer system,were significantly altered in the presence of methyl orange(MO).Moreover,a high concentration of DO heavily impacted the expression levels of genes involved in fatty acid biodegradation.3.Accordingly,we propose a possible pathway for MO biodegradation and discuss the most likely causes of biodegradation inhibition due to dissolved oxygen:(1)azoredctase is likely adjacent to the complex I of the electron transfer chain and(2)azoreduction is a short electron process that overlaps with the initial section of the respiratory electron transfer chain(Fig.6).During MO biodegradation,dioxygen may compete with MO as an electron acceptor.4.The three dimensional structure of azoreductase contains 6 a-helix and 7 β-sheets and adopts a typical Rossmann fold.The active pocket can merely be formed by the homodimer.5.The isoalloxazine ring of FMN is stabilized in planar by residues in the hydraphobic pocket.Hydrogen bonds are formed between FMN and residues Met94,Tyr95,Asn96,Phe97,Gly140,Gly141,Gly143,Thr138,Ser10,Ser16,Gln17 and Ser18 from monomer B.6.Binding of NADH follows a bipartition mode.NADH is superposed against the iaoalloxazine ring with an angle and the distance from C4 atom of NADH to N5 atom of FMN is 4.3 A.The nicotinamide moiety of NADH forms hydrogen bonds with residues Ala111,Tyr119 and Arg59 from monomer A.The adenine nucleotide moiety of NADH forms hydrogen bond with residue Ser16 form monomer B.7.MR is superposed against the iaoalloxazine ring with an angle of 30° and the distance from azo bond of MR to N5 atom of FMN is 4.6 A.Hydrogen bonds are formed between MR and residues Asn96,Gly140 and Gly141from monomer A and residues Tyr119 from monomer B.8.To confirm the predicted binding mode,10 amino acids of soAzoR were separately substituted to generate different mutants.The comparison in kinetic parameters of mutants with wild type suggests that(1)G140F and G141F completely lost the enzymatic activity in degrading MR,(2)the remaining capability in binding both MR and NADH of S16A and N96A was less than 50%,(3)F161A completely lost its enzymatic activity,as well,(4)the NADH binding affinity as well as the enzymatic activity of F117A was greatly improved,(5)the NADH binding affinity of Y119A declined,(6)the MR binding affinity of R139A was at least 4 times lower than the that of the wild type,(7)the remaining activity in degrading MR of H143A was 19%,and(8)the mutant of R59A had littlie impact on the enzymetic acivity of soAzoR.