Characterization and reconstitute of a [Fe4S4] adenosine 5'-phosphosulfate reductase from Acidithiobacillus ferrooxidans.
Adenosine 5'-phosphosulfate (APS) reductase is a key enzyme involved in the pathways of sulfate reduction and sulfide oxidation in the biological sulfur cycle. In this study, the gene of APS reductase from Acidithiobacillus ferrooxidans was cloned and expressed in Escherichia coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. The molecular mass of the recombinant APS reductase was determined to be 28 kDa using SDS-PAGE. According to 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. As we know so far, this is the first report of expression in E. coli of APS reductase from A. ferrooxidans.
Zheng
Chunli
C
School of Resource Processing and Bioengineering, Central South University, Changsha 410083, People's Republic of China.
Zhang
Yanfei
Y
Liu
Yuandong
Y
Wu
Anna
A
Xia
Lexian
L
Zeng
Jia
J
Liu
Jianshe
J
Qiu
Guanzhou
G
D042763:Acidithiobacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C019618:adenylylsulfate reductase