Expression, Purification, And The Characterization Of Rhodanese And Serine Acetyltransferase From Acidithiobacillus Ferrooxidans

Acidithiobacillus ferrooxidans (A. ferrooxidans) is a well known acidophilic, chemolithoautotrophic, Gram negative, bacterium most involved in bioleaching. In aerobic conditions, it gains energy mainly from the oxidation of ferrous iron and/or reduced sulfur compounds present in ores.Rhodaneses is an important enzyme in sulfur oxidation, it catalyzes the sulfane sulfur of thiosulfate to cyanide, formation sulfite and thiocyanate. The biological role of rhodaneses is still largely debated, proposed functions for rhodaneses include cyanide detoxification, formation of prosthetic groups in iron-sulfur cluster proteins, and sulfur transfer for thiamine, thiouridine or molybdopterin biosynthesis. rhodanese domains are present as single or tandem repeats, containing the putative catalytic cysteine in the C-terminal domain, and also as modules in multidomain proteins. Upon analyzing the A. ferrooxidans genome sequence, we found that rhodaneses contained a cysteine motif (Cys-XX-Trp-XX-Cys) at its C-terminal end, known to bind iron-sulfur clusters. The gene of rhodaneses from A. ferrooxidans was cloned, overexpressed and purified by one-step affinity chromatography. The molecular mass of the purified rhodaneses protein was 21KDa. The UV-Vis scanning and EPR spectra results indicated that the wild type proteins contained an iron-sulfur cluster. We constructed the mutant expression plasmid P_rhdA (C92A, C101A, C147A, C197A, C203A) of the protein using site-directed mutagenesis. Site-directed mutagenesis results revealed that the four cycteines Cys92, Cys101, Cys197 and Cys203 were crucial residues for iron-sulfur cluster binding.Serine acetyltransferase (SAT) is a key enzyme in cysteine synthesis, catalyses the formation of O-acetyl-L-serine from acetyl-CoA and L-serine. Cysteine biosynthesis is a key step in the biological sulfur cycle since it is the point at which inorganic sulfur enters into organic combination. The gene of SAT from A. ferrooxidans was cloned, expressed and purified by one-step affinity chromatography. The molecular mass the SAT was 28KDa, SAT have a high enzyme activity in vitro. As we know, this is the first report of expression in Escherichia coli of the SAT from A. ferrooxidans.Three proteins of IscS (a cysteine desulfurase), IscU (a scaffold protein) and IscA (an iron chaperon) encoded by the operon iscSUA were proposed to be involved in the iron-sulfur cluster assembly in A. ferrooxidans, but the mechanism is still not clear so far. Recently, research found that RhdA-SSH can function as sole sulfur source for reconstitution of iron-sulfur clusters in vitro. To confirm this, we used rhodanese to replace the IscS in vitro iron-sulfur cluster assembly. A series contrast results confirmed that rhodanese can catalyses the L-cystine desulfurized, then provided active sulfur in vitro iron-sulfur cluster assembly.