Study Of ATP Sulfurylase, Adenosine 5'-phosphosulfate (APS) Reductase And Cysteine Synthase From Sulfur Assimilation Pathway In Acidithiobacillus Ferrooxidans

In this study, firstly we amplified one of candidate gene cysD-1 of ATP sulfurylase and gene cysH of Adenosine 5'-phosphosulfate (APS) reductase from Acidithiobacillus ferrooxidans which grown at difference media by RT-PCR. The results showed that gene expression quantity and the transcriptional level of the two genes were different.Then the gene of ATPS, APS reductase and cysteine synthase were cloned, expressed and purified. The molecular mass of the recombinant proteins were determined to separately be 33 kDa, 28 kDa and 32 kDa using SDS-PAGE. Colors and UV-vis scanning results of the recombinant ATPS confirmed that pyridoxal 5-phosphate (PLP) inserted into the active site of the protein. We first discovered ATPS containing PLP cofactor.Purified APS reductase showed brown-yellow. Optical and EPR spectra results of the recombinant protein confirmed that the iron-sulfur cluster inserted into the active site of the protein. Site-directed mutation for the enzyme revealed that Cys110, Cys111, Cys193 and Cys196 were in ligation with the iron-sulfur cluster. The [Fe₄S₄] cluster could be assembled in vitro, and exhibited electron transport and redox catalysis properties.Purified cysteine synthase showed light-yellow. Colors and UV-vis scanning results of the recombinant protein confirmed that pyridoxal 5'-phosphate (PLP) inserted into the active site of the protein. Fluoresence spectrum showed energy transfer from tryptophan to the enolimine tautomer of the internal aldimine. Alignment sequences and site-directed mutation for the enzyme revealed that K30 and H150 and H168 were crucial residues for PLP binding. Measurement of the enzyme activity indicates which can catalyze sulfide and O-acetylserine (OAS) formation of cysteine.The results are consistent with our previous studies, which proved that a Sulfur uptake and assimilation pathway exists in A. ferrooxidans. Firstly, sulfate is subjected to activation to adenosine 5'-phosphosulfate (5'-adenylylsulfate [APS]). Then APS is reduced to sulfite (SO₃^2-) and then sulfide (S^2-). Sulfide is then coupled with O-acetyl-Ser (OAS) that is formed from Ser, yielding Cysteine.