Expression, Purification And Exzymatic Characteristics Of Cysteine Desulfurase, IscS From Acidithiobacillus Ferrooxidans

Acidithiobacillus ferrooxidans(A.ferrooxidans) is one of the most studied microorganisms commonly found in bioleaching and acid mine drainage.Understanding the respiration of iron and reduced sulfur compounds is of wide interest,not only scientifically,but also from an economic and environmental point of view.To gain further insight into the physiological role of the known redox proteins and to help identify yet unknown or missing components involved in iron and sulfur respiratory chains to oxygen,the cysteine desulfurase IscS encoded by genes in genome ofA.ferrooxidans was studied.The gene of IscS from Acidithiobacillus ferrooxidans ATCC 23270 was cloned,successfully expressed in Escherichia coli,and purified by one-step affinity chromatography to homogeneity.The purity of the IscS and its mutant proteins was further examined by SDS-PAGE and single bands corresponding to the 46 kDa.The wild-type IscS protein had an intense yellow color.The UV-visible spectra of the recombinant IscS indicated a major absorption peak at 416 nm,which is typical for proteins containing a pyridoxal 5'-phosphate(PLP) cofactor.The IscS catalyzes the elimination of S from L-cysteine to yield L-alanine and elemental sulfur,provided sulfur for the assembly of iron-sulfur cluster. Site-directed mutagenesis and the modeled overall structure for the IscS revealed that His106,Lys208,Ser329,Cys331 and Leu336 were involved in PLP binding and its activity.The optimum reaction temperature of cysteine desulfurase was 30℃, the optimum reaction pH was 8.0-8.5.The enzyme was stable at 35℃but was unstable at temperatures above 55℃.1,5-I-AEDANS signify-cantly inhibited IscS activity.Kinetic parameters K_m and V_max were found to be 0.11 mmol/L and 2.57μmol/(L.min).The[Fe₂S₂]cluster assembly in target protein in vivo required scaffold proteins of IscA,IscS and IscU,which might follow the Isc "USA" pathway.The results suggests that the cysteine desulfurase,IscS,plays a major role in Fe-S cluster formation in E.coli,inactivation of IscS seriously affected the iron-sulfur cluster assembly in E.coli.