Structure Model And Function Of Azoreductase AZR

Biodegradation of azo dyes has attracted a lot of attention in recent years.Many azo-dye-degrading microorganisms were isolated and characterized.In addition,several efficient azoreductases were obtained and studied.However,in-depth understanding of the structure and function of azoreductase is limited.Bacterial decolorization of azo dyes and the structure model and function of FMN-dependent azoreductase are studied in this dissertation.Decolorization of azo dyes by Rhodopseudomonas palustris AS1.2352 and its cell extracts were investigated.Under anoxic conditions,the growing strain could decolorize over 80%of 50 mg L^-1 reactive brilliant red X-3B(RBR X-3B) in 17 h.The optimal temperature and pH were around 30-35℃and pH 6-9,respectively.The correlation between specific decolorization rate and dye concentration could be described by Michaelis-Menten kinetics. The kinetic constants estimated were 65 mg g cell^-1 h^-1 for V_max and 978 mg L^-1 for K_m. Azoreductase activity was found in its cell extracts,which demonstrated more effective decolorization performance than whole cells.Decolorization of sulfonated azo dyes was investigated with early-stationary-phase cells of two photosynthetic bacteria(PSB, Rhodobacter sphaeroides AS1.1737 and R.palustris AS1.2352) and a gene-engineered strain (Escherichia coli YB).The two PSB strains demonstrated higher decolorization abilities than the recombinant one.Values of V_max/K_m were 0.2,0.15 and 0.06 L g cell^-1 h^-1 for R. sphaeroides AS1.1737,R.palustris AS1.2352 and E.coli YB,respectively.The two PSB strains showed higher decolorization abilities for mixed azo dyes than E.coli YB.Structure model of azoreductase AZR of R.sphaeroides AS 1.1737 was constructed using homology modeling method.AZR monomer demonstrates a flavodoxin-like globularα/βstructure with the central five-stranded parallelβ-sheet flanked by fiveαhelices.An FMN prosthetic group which lies on top of the molecule may act as redox and reactive centers. Although FMN-dependent azoreductases can be classified into two goups according to amino acid sequence,their monomers demonstrated similar flavodoxin-like 3D structures.Many nitroaromatics could be reduced by AZR using NADPH as electron donor.The optimal temperature and pH for its nitroreductase activity was 50℃and pH 7,respectively. Compared with NADH,NADPH was a better electron donor.The reaction followed Ping-Pong Bi Bi kinetic mechanism.It was demonstrated that,among 2,4,6-trinitrotoluene (TNT),2,4-dinitrotoluene(2,4-DNT) and 2,6-dinitrotoluene(2,6-DNT),TNT was the best substrate and 2,4-DNT was a better substrate than 2,6-DNT.Hydroxylamino-dinitrotoluene was detected with HPLC/MS as reduction products of TNT.AZR could also reduce FMN following Ping-Pong Bi Bi kinetic mechanism.K_m values for NADPH and FMN were 0.5 and 14.2 mM,respectively.V_max was calculated to be 172.4μmol mg protein^-1 min^-1.Externally added FMN was a competitive inhibitor of NADPH,methyl red and nitrofurazone.The inhibition constants for NADPH,methyl red and nitrofurazone were 1.7,2.3 and 6.4μM, respectively.AZR has a wide range of substrates,including azo and nitro compounds,FMN and metal ions.It is a member of a new nitro-/FMN reductase family.On the basis of model analysis and multiple sequence alignment,several amino acid residues(Tyr⁷⁴,His⁷⁵ and Lys¹⁰⁹) in the two loops around FMN were chosen as mutation targets.Site-directed mutagenesis including K109A,K109H,Y74W and H75N were performed with multiple steps of PCR.Wild-type and mutant AZR proteins were heterogeously overexpressed in E.coli and purified before characterization.Using acid red B as substrate,the optimal pH value of wild type AZR was pH 7-8.While those of K109H and H75N were pH 6,and those of K109A and Y74W were pH 9.The mutations might alter the surficial charge of AZR and resulted in the variation of optimal pH values.Compared to the wild-type AZR,decreased activities were found with all the mutant ones,which indicated that K¹⁰⁹,Y⁷⁴ and H⁷⁵ are essential residues for the activity of AZR.According to the analysis of kinetic parameters,the following conclusions were obtained.Positively charged residues at the 109^th site is necessary for the binding of methyl red,but not for nitrofurazone.Mutations of Y74W and H75N do not affect the binding of NADPH,however they decrease the binding of methyl red and nitrofurazone.The H75N mutant loses its nitroreductase activity completely. K109H is not a conserved mutation for the binding of NADPH,which may also be affected by local spatial structures.K¹⁰⁹ may only be involved in the binding of the 2'-phosphate group of NADPH and have no effect on the binding of NADH.There are very few natural azo and nitro aromatics,thus AZR may have some other functions rather than azo- or nitro- reductase.Quinone compounds are widly found in natural environments and metabolism processes of many different lives.A short partial sequence (40-50 amino acid residues) involved in the binding of substrate and coenzyme was found conserved among AZR and several other quinone reductases from different domains.Similarα/βmonomer structures and ways of flavin cofactors binding were found between AZR and quinone reductase NQO1.It was demonstrated that AZR is a two-electron quinone reductase following the Ping-Pong Bi Bi kinetics.It could reduce 2-methyl-1,4-naphthoquinone (menadione),2-hydroxy-1,4-naphthoquinone(lawsone,LQ),anthraquinone-2-sulfonate and anthraquinone-2,6-disulfonate.Menadione was the best substrate among quinones investigated.No activity was detected with 1,4-benzoquinone.Compared to azo and nitro compounds,quinones are better substrate of AZR.The quinone reductase activity of AZR was inhibited by dicoumarol,which was a competitive inhibitor of NADPH with a K_i of 87.6μM. The overexpressed cellular quinone reductase AZR significantly increased the resistance of E. coli YB to oxidative stress.When treated with stressors such as H₂O₂,menadione,paraquat and heat shock,the survival rates of E.coli YB were higher than those of the control strain E. coli JM109.The addition of redox mediator LQ significantly enhanced the decolorization performances of E.coli JM109 and E.coli YB.The recombinant strain possessed better mediated decolorization ability than the control one.In the presence of 0.2 mM LQ,E.coli YB removed 75%amaranth(1 mM) in 2 h,which is the best bacterial decolorization performance ever reported.This demonstrated the advantage of gene-engineered strain and provided a new strategy for the application of heterogeneously expressed intracellular reductase.The study provides a deeper understanding of the structure and function of FMN-dependent azoreductase.It lays the foundation for the application of AZR and other reductases.